Pyrazolopyrimidine compounds

ABSTRACT

Compounds of formula (I) and salts thereof: 
                         
wherein R 1  is n-C 1-6 alkyl or C 1-2 alkoxyC 1-2 alkyl-, R 2  is halo, OH or C 1-3 alkyl, m is an integer having a value of 4, 5, 6 or 7, n is an integer having a value of 0, 1, 2 or 3, and p is an integer having a value of 0, 1 or 2, are inducers of human interferon. Compounds which induce human interferon may be useful in the treatment of various disorders, for example the treatment of allergic diseases and other inflammatory conditions, for example allergic rhinitis and asthma, infectious diseases and cancer, and may also be useful as vaccine adjuvants.

FIELD OF THE INVENTION

The present invention relates to compounds, processes for theirpreparation, compositions containing them, to their use in the treatmentof various disorders in particular allergic diseases and otherinflammatory conditions for example allergic rhinitis and asthma,infectious diseases, and cancer, and as vaccine adjuvants.

BACKGROUND OF THE INVENTION

Vertebrates are constantly threatened by the invasion of microorganismsand have evolved mechanisms of immune defense to eliminate infectivepathogens. In mammals, this immune system comprises two branches; innateimmunity and acquired immunity. The first line of host defense is theinnate immune system, which is mediated by macrophages and dendriticcells. Acquired immunity involves the elimination of pathogens at thelate stages of infection and also enables the generation ofimmunological memory. Acquired immunity is highly specific, due to thevast repertoire of lymphocytes with antigen-specific receptors that haveundergone gene rearrangement.

Central to the generation of an effective innate immune response inmammals are mechanisms which bring about the induction of interferonsand other cytokines which act upon cells to induce a number of effects.In man, the type I interferons are a family of related proteins encodedby genes on chromosome 9 and encoding at least 13 isoforms of interferonalpha (IFNα) and one isoform of interferon beta (IFNβ). Interferon wasfirst described as a substance which could protect cells from viralinfection (Isaacs & Lindemann, J. Virus Interference. Proc. R. Soc. Lon.Ser. B. Biol. Sci. 1957: 147, 258-267). Recombinant IFNα was the firstapproved biological therapeutic and has become an important therapy inviral infections and in cancer. As well as direct antiviral activity oncells, interferons are known to be potent modulators of the immuneresponse, acting on cells of the immune system (Gonzalez-Navajas J. M.et at Nature Reviews Immunology, 2012; 2, 125-35).

Toll-like receptors (TLRs) are a family of ten Pattern RecognitionReceptors described in man (Gay, N. J. et al, Annu. Rev. Biochem., 2007:46, 141-165). TLRs are expressed predominantly by innate immune cellswhere their role is to monitor the environment for signs of infectionand, on activation, mobilize defense mechanisms aimed at the eliminationof invading pathogens. The early innate immune-responses triggered byTLRs limit the spread of infection, while the pro-inflammatory cytokinesand chemokines that they induce lead to recruitment and activation ofantigen presenting cells, B cells, and T cells. The TLRs can modulatethe nature of the adaptive immune-responses to give appropriateprotection via dendritic cell-activation and cytokine release (Akira S.et al, Nat. Immunol., 2001: 2, 675-680). The profile of the responseseen from different TLR agonists depends on the cell type activated.

TLR7 is a member of the subgroup of TLRs (TLRs 3, 7, 8, and 9),localised in the endosomal compartment of cells which have becomespecialised to detect non-self nucleic acids. TLR7 plays a key role inanti-viral defense via the recognition of ssRNA (Diebold S. S. et al,Science, 2004: 303, 1529-1531; and Lund J. M. et al, PNAS, 2004: 101,5598-5603). TLR7 has a restricted expression-profile in man and isexpressed predominantly by B cells and plasmacytoid dendritic cells(pDC), and to a lesser extent by monocytes. Plasmacytoid DCs are aunique population of lymphoid-derived dendritic cells (0.2-0.8% ofPeripheral Blood Mononuclear Cells (PBMCs)) which are the primary type Iinterferon-producing cells secreting high levels of interferon-alpha(IFNα) and interferon-beta (IFNβ) in response to viral infections (LiuY-J, Annu. Rev. Immunol., 2005: 23, 275-306).

Administration of a small molecule compound which could stimulate theinnate immune response, including the activation of type I interferonsand other cytokines via Toll-like receptors, could become an importantstrategy for the treatment or prevention of human diseases. Smallmolecule agonists of TLR7 have been described which can induceinterferon alpha in animals and in man (Takeda K. et al, Annu. Rev.Immunol., 2003: 21, 335-76). TLR7 agonists include imidazoquinolinecompounds such as imiquimod and resiquimod, oxoadenine analogues andalso nucleoside analogues such as loxoribine and 7-thia-8-oxoguanosinewhich have long been known to induce interferon alpha (Czarnecki. M., J.Med, Chem., 2008: 51, 6621-6626; Hedayat M. et al, Medicinal ResearchReviews, 2012: 32, 294-325). This type of immunomodulatory strategy hasthe potential to identify compounds which may be useful in the treatmentof allergic diseases (Moisan J. et al, Am. J. Physiol. Lung Cell Mol.Physiol., 2006: 290, L987-995), viral infections (Horcroft N.J. et al,J. Antimicrob. Chemther, 2012: 67, 789-801), cancer (Krieg A., Curr.Oncol. Rep., 2004: 6(2), 88-95), other inflammatory conditions such asirritable bowel disease (Rakoff-Nahoum S., Cell., 2004, 23, 118(2):229-41), and as vaccine adjuvants (Persing et al. Trends Microbiol.2002: 10(10 Supply, 532-7).

More specifically, allergic diseases are associated with a Th2-biasedimmune-response to allergens. Th2 responses are associated with raisedlevels of IgE, which, via its effects on mast cells, promotes ahypersensitivity to allergens, resulting in the symptoms seen, forexample, in asthma and allergic rhinitis. In healthy individuals theimmune-response to allergens is more balanced with a mixed Th2/Th1 andregulatory T cell response. TLR7 ligands have been shown to reduce Th2cytokine and enhance Th1 cytokine release in vitro and to ameliorateTh2-type inflammatory responses in allergic lung models in vivo (DuechsM. J., Pulmonary Pharmacology & Therapeutics, 2011: 24, 203-214; Ali L.et al, J. All. Clin. Immunol., 2006: 118, 511-517; Tao et al, Chin. Med.J., 2006: 119, 640-648; Van L. P. Eur. J. Immunol, 2011: 41, 1992-1999).Thus TLR7 ligands have the potential to rebalance the immune-responseseen in allergic individuals and lead to disease modification. Recentclinical studies with the TLR7 agonist have shown repeated intranasalstimulation of TLR7 to produce a sustained reduction in theresponsiveness to allergen in patients with both allergic rhinitis andallergic asthma (Greiff L. Respiratory Research, 2012: 13, 53; Leaker B.R. et al, Am. J. Respir. Crit. Care Med., 2012: 185, A4184).

In the search for novel small molecule inducers of human interferon IFNαan assay strategy has been developed to characterise small molecule(regardless of mechanism) which is based on stimulation of primary humandonor cells or whole blood with compounds, and is disclosed herein.

SUMMARY OF THE INVENTION

In a first aspect, the present invention is directed to compounds offormula (I) and salts thereof:

wherein:

-   R₁ is n-C₁₋₆alkyl or C₁₋₂alkoxyC₁₋₂alkyl-;-   R₂ is halo, hydroxy or C₁₋₃alkyl;-   m is an integer having a value of 4 to 7;-   n is an integer having a value of 0 to 3;-   p is an integer having a value of 0 to 2.

Certain compounds of the invention have been shown to be inducers ofhuman interferon and may possess a desirable developability profilecompared to known inducers of human interferon. In addition, certaincompounds of the invention may also show selectivity for IFNα withrespect to TNFα. Compounds which induce human interferon may be usefulin the treatment of various disorders, for example the treatment ofallergic diseases and other inflammatory conditions, for exampleallergic rhinitis and asthma, the treatment of infectious diseases andcancer. Accordingly, the invention is further directed to pharmaceuticalcompositions comprising a compound of formula (I), or a pharmaceuticallyacceptable salt thereof. The present invention is further directed tomethods of treatments of disorders associated therewith using a compoundof formula (I) or a pharmaceutically acceptable salt thereof, or apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof.

The compounds of the invention may also have use as vaccine adjuvants.Consequently, the present invention is further directed to a vaccinecomposition comprising a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, and an antigen or antigen composition.

Certain compounds of the invention are potent immunomodulators andaccordingly, care should be exercised in their handling.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 illustrates the XRPD diffractogram of Example 21.

FIG. 2 illustrates the XRPD diffractogram of Example 22.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention is directed to compounds offormula (I) and salts thereof:

wherein:

-   R₁ is n-C₁₋₆alkyl or C₁₋₂alkoxyC₁₋₂alkyl-;-   R₂ is halo, hydroxy or C₁₋₃alkyl;-   m is an integer having a value of 4 to 7;-   n is an integer having a value of 0 to 3;-   p is an integer having a value of 0 to 2.

In a further aspect, the present invention is directed to compounds offormula (I) and salts thereof:

wherein:

-   R₁ is n-C₃₋₆alkyl or C₁₋₂alkoxyC₁₋₂alkyl-;-   each R₂ independently represents halo, OH or C₁₋₃alkyl;-   m is an integer having a value of 4, 5, 6 or 7;-   n is an integer having a value of 0, 1, 2 or 3;-   p is an integer having a value of 0, 1 or 2.

In a further aspect, the present invention is directed to compounds offormula (I) and salts thereof:

wherein:

-   R₁ is n-C₄₋₆alkyl;-   R₂ is halo or OH;-   m is an integer having a value of 5, 6 or 7;-   n is an integer having a value of 1, 2 or 3;-   p is an integer having a value of 0 or 1.

In a further aspect, the present invention is directed to compounds offormula (I) and salts thereof:

wherein:

-   R₁ is n-butyl or 2-methoxyethyl;-   R₂ is halo or OH;-   m is an integer having a value of 5, 6 or 7;-   n is an integer having a value of 1, 2 or 3;-   p is an integer having a value of 0 or 1.

In a further aspect, the present invention is directed to compounds offormula (I) and salts thereof:

wherein:

-   R₁ is n-butyl or 2-methoxyethyl;-   R₂ is F or OH;-   m is an integer having a value of 5, 6 or 7;-   n is an integer having a value of 1, 2 or 3;-   p is an integer having a value of 0 or 1.

In a further aspect, the present invention is directed to compounds offormula (I) and salts thereof:

wherein:

-   R₁ is n-butyl or 2-methoxyethyl;-   m is an integer having a value of 5, 6 or 7;-   n is an integer having a value of 1, 2 or 3;-   p is 0.-   In a further aspect, R₁ is n-C₄₋₆alkyl, for example n-butyl.-   In a further aspect, R₁ is 2-methoxyethyl.-   In a further aspect, m is an integer having a value of 5 or 6.-   In a further aspect, n is 1 or 2.-   In a further aspect, p is 0 or 1.-   In a further aspect, R₂ is halo or OH.-   In a further aspect, R₂ is F or OH.

Examples of compounds of formula (I) are provided in the followinggroup, and form a further aspect of the invention:

-   5-Butyl-3-(6-(piperidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine;-   5-(2-Methoxyethyl)-3-(6-(piperidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine;-   5-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine;-   5-(2-Methoxyethyl)-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine;-   5-Butyl-3-(5-(piperidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine;-   5-Butyl-3-(5-(pyrrolidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine;-   5-Butyl-3-(7-(piperidin-1-yl)heptyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine;-   5-Butyl-3-(7-(pyrrolidin-1-yl)heptyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine;-   3-(6-(Azepan-1-yl)hexyl)-5-butyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine;-   3-(5-(Azepan-1-yl)pentyl)-5-butyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine;-   (S)-5-Butyl-3-(6-(3-fluoropyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine;-   (S)-5-Butyl-3-(5-(3-fluoropyrrolidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine;-   (R)-5-Butyl-3-(6-(3-fluoropyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine;-   (R)-5-Butyl-3-(5-(3-fluoropyrrolidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine;-   1-(6-(7-Amino-5-butyl-1H-pyrazolo[4,3-d]pyrimidin-3-yl)hexyl)piperidin-4-ol;-   1-(5-(7-Amino-5-butyl-1H-pyrazolo[4,3-d]pyrimidin-3-yl)pentyl)piperidin-4-ol;-   5-Butyl-3-(6-(4-fluoropiperidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine;-   5-Butyl-3-(5-(4-fluoropiperidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine,    and salts thereof.

In a further aspect, the present invention is directed to5-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine,or a salt thereof.

As used herein, the term “alkyl” refers to a saturated, hydrocarbonchain having the specified number of member atoms. Unless otherwisestated, the term ‘alkyl’ includes straight and branched alkyl groups.For example, C₁₋₆alkyl refers to a saturated, straight or branchedhydrocarbon chain having from 1 to 6 carbon atoms, such as ethyl andisopropyl, and n-C₁₋₆alkyl refers to a saturated, straight hydrocarbonchain having from 1 to 6 carbon atoms, such as n-propyl, and n-butyl.

It is to be understood that references herein to compounds of theinvention mean a compound of formula (I) as the free base, or as a salt,for example a pharmaceutically acceptable salt.

In one aspect of the invention, a compound of formula (I) is in the formof a free base.

Salts of the compounds of formula (I) include pharmaceuticallyacceptable salts and salts which may not be pharmaceutically acceptablebut may be useful in the preparation of compounds of formula (I) andpharmaceutically acceptable salts thereof.

In one aspect of the invention, a compound of formula (I) is in the formof a pharmaceutically acceptable salt.

Salts may be derived from certain inorganic or organic acids.

As used herein, the term “pharmaceutically-acceptable salts” refers tosalts that retain the desired biological activity of the subjectcompound and exhibit minimal undesired toxicological effects. Thesepharmaceutically-acceptable salts may be prepared in situ during thefinal isolation and purification of the compound, or by separatelyreacting the purified compound in its free acid or free base form with asuitable base or acid, respectively. Furthermore,pharmaceutically-acceptable salts of the compound of formula (I) may beprepared during further processing of the free acid or base form, forexample in situ during manufacture into a pharmaceutical formulation.

Examples of salts are pharmaceutically acceptable salts.Pharmaceutically acceptable salts include acid addition salts. For areview on suitable salts see Berge et al., J. Pharm. Sci., 66:1-19(1977).

Examples of pharmaceutically acceptable acid addition salts of acompound of formula (I) include inorganic acids such as, for example,hydrochloric acid, hydrobromic acid, orthophosphoric acid, nitric acid,phosphoric acid, or sulphuric acid, or with organic acids such as, forexample, methanesulphonic acid, ethanesulphonic acid, p-toluenesulphonicacid, acetic acid, propionic acid, lactic acid, citric acid, fumaricacid, malic acid, succinic acid, salicylic acid, maleic acid,glycerophosphoric acid, tartaric, benzoic, glutamic, aspartic,benzenesulphonic, naphthalenesulphonic such as 2-naphthalenesuphonic,hexanoic acid or acetylsalicylic acid.

The invention includes within its scope all possible stoichiometric andnon-stoichiometric forms of the salts of the compounds of formula (I).For example, a dimaleate or hemi-succinate salt of the compound offormula (I).

Salts may be formed using techniques well-known in the art, for exampleby precipitation from solution followed by filtration, or by evaporationof the solvent.

Typically, a pharmaceutically acceptable acid addition salt can beformed by reaction of a compound of formula (I) with a suitable acid(such as hydrobromic, hydrochloric, sulphuric, maleic,p-toluenesulphonic, methanesulphonic, naphthalenesulphonic or succinicacids), optionally in a suitable solvent such as an organic solvent, togive the salt which is usually isolated for example by crystallisationand filtration.

Examples of pharmaceutically acceptable salts of5-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amineare the maleate, dimaleate, and hemi-succinate salts.

It will be appreciated that many organic compounds can form complexeswith solvents in which they are reacted or from which they areprecipitated or crystallised. These complexes are known as “solvates”.For example, a complex with water is known as a “hydrate”. Solvents withhigh boiling points and/or solvents with a high propensity to formhydrogen bonds such as water, ethanol, iso-propyl alcohol, and N-methylpyrrolidinone may be used to form solvates. Methods for theidentification of solvated include, but are not limited to, NMR andmicroanalysis. Solvates of the compounds of formula (I) are within thescope of the invention. As used herein, the term solvate encompassessolvates of both a free base compound as well as any salt thereof.

Certain of the compounds of the invention may exist in tautomeric forms.It will be understood that the present invention encompasses all of thetautomers of the compounds of the invention whether as individualtautomers or as mixtures thereof.

The compounds of the invention may be in crystalline or amorphous form.Furthermore, some of the crystalline forms of the compounds of theinvention may exist as polymorphs, all of which are included within thescope of the present invention. The most thermodynamically stablepolymorphic form or forms of the compounds of the invention are ofparticular interest.

In a further aspect, the present invention is directed to a crystallinesolid state form of5-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-aminedimaleate, characterised by an X-ray powder diffraction pattern havingdiffraction peaks at 26 values of 5.3, 5.8, 6.4, 9.0, 10.1, 10.9, 11.6,12.7, 16.0 and 19.1.

In a further aspect, the present invention is directed to a crystallinesolid state form of5-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-aminehemi-succinate, characterised by an X-ray powder diffraction patternhaving diffraction peaks at 26 values of 8.1, 9.8, 11.6, 16.0, 17.5,19.5, 20.2, 23.0 and 23.7.

Polymorphic forms of compounds of the invention may be characterised anddifferentiated using a number of conventional analytical techniques,including, but not limited to, X-ray powder diffraction (XRPD), infraredspectroscopy (IR), Raman spectroscopy, differential scanning calorimetry(DSC), thermogravimetric analysis (TGA) and solid-state nuclear magneticresonance (ssNMR).

The present invention also includes all suitable isotopic variations ofa compound of formula (I) or a pharmaceutically acceptable salt thereof.An isotopic variation of a compound of formula (I), or apharmaceutically acceptable salt thereof, is defined as one in which atleast one atom is replaced by an atom having the same atomic number butan atomic mass different from the atomic mass usually found in nature.Examples of isotopes that can be incorporated into compounds of theinvention include isotopes of hydrogen, carbon, nitrogen, oxygen,fluorine and chlorine such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ¹⁸F and³⁶Cl, respectively. Certain isotopic variations of a compound of formula(I) or a salt or solvate thereof, for example, those in which aradioactive isotope such as ³H or ¹⁴C is incorporated, are useful indrug and/or substrate tissue distribution studies. Tritiated, i.e., ³H,and carbon-14, i.e., ¹⁴C, isotopes are particularly preferred for theirease of preparation and detectability. Further, substitution withisotopes such as deuterium, i.e., ²H, may afford certain therapeuticadvantages resulting from greater metabolic stability, for example,increased in vivo half-life or reduced dosage requirements and hence maybe preferred in some circumstances. Isotopic variations of a compound offormula (I), or a pharmaceutically salt thereof, can generally beprepared by conventional procedures such as by the illustrative methodsor by the preparations described in the Examples hereafter usingappropriate isotopic variations of suitable reagents.

It will be appreciated from the foregoing that included within the scopeof the invention are solvates, hydrates, isomers, isotopic variationsand polymorphic forms of the compounds of formula (I) and salts andsolvates thereof.

Compound Preparation

The compounds of formula (I) and salts thereof may be prepared by themethodology described hereinafter, constituting further aspects of thisinvention.

Accordingly, there is provided a process for the preparation of acompound of formula (I), which process comprises the functional groupinterconversion or deprotection of a compound of formula (II):

wherein R₁, R₂, m, n and p are as defined hereinbefore for a compound offormula (I) and Z is OH or amino substituted with a suitable protectinggroup such as 3,4-dimethoxybenzyl or 2,4-dimethoxybenzyl and thereafter,if required, preparing a salt of the compound so-formed.

For example when Z is OH, a compound of formula (II) is dissolved inphosphorus oxychloride and heated, at a suitable temperature, forexample 120° C. for a suitable period, for example 45-120 minutes. Thereaction mixture is evaporated and azeotroped with a suitable solvent,for example toluene. A solution of aqueous ammonia (0.88) is then addedto a solution of the material in a suitable solvent, for exampleiso-propyl alcohol. The resultant mixture is then heated in a microwaveheater at a suitable temperature, for example 120-150° C. for a suitableperiod of time, for example 1-2 hours. The product (I) is isolated byremoval of the solvent and purification if required.

For example when Z is a (3,4-dimethoxyphenyl)methanamine group, acompound of formula (II) is treated with a suitable acid such astrifluoroacetic acid and heated at a suitable temperature, for example120° C., in a microwave heater for a suitable period of time, forexample 4 hours. The product (I) is isolated by removal of the solvent,aqueous work up and purification if required.

For example when Z is a (2,4-dimethoxyphenyl)methanamine group, acompound of formula (II) is treated with a suitable acid such astrifluoroacetic acid and heated at a suitable temperature, for example60° C. for a suitable period of time, for example 2.5-4 hours. Theproduct (I) is isolated by the removal of solvent, aqueous work-up andpurification if required.

A compound of formula (II) may be prepared by reaction of a compound offormula (III):

wherein R₁, R₂, m, n and p are as hereinbefore defined for a compound offormula (I) and Z is as hereinbefore defined for a compound of formula(II), with hydrogen in the presence of a catalyst.

For example a compound of formula (III) is dissolved in a suitablesolvent for example ethyl alcohol, and passed over a suitable catalyst,for example 10% palladium on carbon, in the presence of hydrogen at asuitable temperature, for example 20-60° C., in a suitable flowhydrogenation apparatus such as the Thales H-Cube™ Alternatively acompound of formula (III) is dissolved in a suitable solvent for exampleethyl alcohol and stirred under an atmosphere of hydrogen in thepresence of a suitable catalyst, for example 10% palladium on carbon, ata suitable temperature, for example 20° C., for a suitable period oftime 2-18 hours. The product (II) is isolated by removal of the solvent,aqueous work up and purification if required.

A compound of formula (III) may be prepared by reaction of a compound offormula (IV):

wherein R₁ is as hereinbefore defined for a compound of formula (I), Zis as hereinbefore defined for a compound of formula (II) and X is ahalogen, such as iodine or bromine, with a compound of formula (V):

wherein R₂, m, n and p are defined for a compound of formula (I).

For example a compound of formula (IV), is dissolved in a suitablesolvent, for example N,N-dimethylformamide, in the presence of copper(I)iodide, a suitable catalyst, for examplebis(triphenylphosphine)palladium(II) dichloride and a suitable base, forexample triethylamine. A solution of a compound of formula (V) in asuitable solvent, such as N,N-dimethylformamide, is added and themixture stirred at a suitable temperature, for example 20-55° C. for asuitable period of time, for example 0.5-17 hours. The product (III) isisolated after an aqueous work-up and purification.

A compound of formula (V) may be prepared by reaction of a compound offormula (VI):

wherein m is defined for a compound of formula (I) and Y is a leavinggroup such as a halogen, for example chlorine, bromine or iodine, or analkyl sulfonate, for example p-toluenesulfonate, with a compound offormula (VII):

wherein R₂, n and p are as defined for a compound of formula (I).

For example a compound of formula (VI), a compound of formula (VII) anda suitable base, for example sodium hydrogen carbonate, are dissolved ina suitable solvent, for example N,N-dimethylformamide, and heated at asuitable temperature, for example 80-100° C. for a suitable period oftime, for example 16-18 hours. The product (V) is isolated after aqueouswork-up and purification, for example by isolation of a suitablecrystalline salt, for example the oxalate salt.

Alternatively a compound of formula (III) may be prepared by reaction ofa compound of formula (VIII):

wherein R₁ and m are as hereinbefore defined for a compound of formula(I), Z is as hereinbefore defined for a compound of formula (II) and Yis a leaving group as defined for compounds of formula (VI), with acompound of formula (VII) wherein R₂, n and p are as defined for acompound of formula (I).

For example a compound of formula (VIII), a compound of formula (VII)and a suitable base, for example triethylamine, are dissolved in asuitable solvent, for example acetontrile and heated at a suitabletemperature, for example 60-80° C. for a suitable period of time, forexample 16-26 hours. The product (III) is isolated after an aqueouswork-up and purification.

Compounds of formula (VIII) can be prepared by reaction of compounds offormula (IV) with compounds of formula (VI). For example, a compound offormula (IV) is dissolved in a suitable solvent, for exampleN,N-dimethylformamide, in the presence of copper(I) iodide, a suitablecatalyst, for example bis(triphenylphosphine)palladium(II) dichlorideand a suitable base, for example triethylamine. A solution of a compoundof formula (VI) in a suitable solvent, such as N,N-dimethylformamide, isadded and the mixture is stirred at a suitable temperature, for example20-60° C. for a suitable period of time, for example 2-18 hours. Theproduct (VIII) is isolated after an aqueous work-up and purification.

Alternatively a compound of formula (II) may be prepared by reaction ofcompounds of formula (IX):

wherein R₁ and m are as hereinbefore defined for a compound of formula(I), Z is as hereinbefore defined for a compound of formula (II) and Xis a leaving group such as a halogen, for example chloro, bromo or iodo,with a compound of formula (VII) wherein R₂, n and p are as defined fora compound of formula (I).

For example a mixture of compound of formula (IX), a compound of formula(VII) and a suitable base, for example triethylamine, in a suitablesolvent, for example acetonitrile, is stirred at a suitable temperature,for example 20° C., for a suitable period of time, for example 17-19hours. The product (IX) is isolated after an aqueous work-up andpurification.

Compounds of formula (IX) may be prepared by the reaction of compoundsof formula (X):

wherein R₁ and m are as hereinbefore defined for a compound of formula(I), Z is as hereinbefore defined for a compound of formula (II) with asuitable halogenating reagent.

For example a solution of triphenylphosphine in a solvent, for exampledichloromethane, in added to a mixture of compound of formula (X) andcarbon tetrabromide in a suitable solvent, for example, dichloromethane.The reaction is stirred as a suitable temperature, for example 20° C.,for a suitable period of time, 18-20 hours. The product (X) is isolatedafter an aqueous work-up and purification.

Compounds of formula (X) may be prepared by reaction of compounds offormula (XI):

wherein R₁ and m are as hereinbefore defined for a compound of formula(I), Z is as hereinbefore defined for a compound of formula (II) withhydrogen in the presence of a catalyst.

For example a compound of formula (XI) is dissolved in a suitablesolvent, for example ethanol, and stirred under an atmosphere ofhydrogen in the presence of a suitable catalyst, for example 10%palladium on carbon, for a suitable period of time, for example 22hours. The product (XIV) is isolated by removal of the solvent andpurification if required.

Compounds of formula (XI) can be prepared by reaction of compounds offormula (IV) with appropriate alkyn-1-ols. For example, a compound offormula (IV) is dissolved in a suitable solvent, for exampleN,N-dimethylformamide, in the presence of copper(I) iodide, a suitablecatalyst, for example bis(triphenylphosphine)palladium(II) dichlorideand a suitable base, for example triethylamine. A solution of thealkyn-1-ol in a suitable solvent, such as N,N-dimethylformamide, isadded and the mixture is stirred at a suitable temperature, for example60° C. for a suitable period of time, for example 2-4 hours. The product(XI) is isolated after an aqueous work-up and purification.

Compounds of formula (IV) wherein Z is an amino substituted with asuitable protecting group can be prepared from compounds of formula (IV)wherein Z is an OH group. For example a compound of formula (IV) Z═OH isdissolved in a suitable solvent, for example N,N-dimethylformamide, inthe presence of a suitable coupling agent, for example(benzotriazol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate, and a base, for example,1,8-diazabicyclo[5.4.0]undec-7-ene, is treated with a suitable amine,for example, (3,4-dimethoxyphenyl)methanamine. The reaction is stirredas a suitable temperature, for example 40° C., for a suitable period oftime, for example 3 hours. The product (IV) where Z is(3,4-dimethoxyphenyl)methanamine is isolated after an aqueous work-upand purification.

Alternatively a compound of formula (IV) Z═OH is dissolved in a suitablesolvent, for example acetonitrile, in the presence of a suitablecoupling agent, for example((1H-benzo[d][1,2,3]triazol-1-yl)oxy)tri(pyrrolidin-1-yl)phosphoniumhexafluorophosphate(V), and a base, for example,1,8-diazabicyclo[5.4.0]undec-7-ene, is treated with a suitable amine,for example, (2,4-dimethoxyphenyl)methanamine. The reaction is stirredas a suitable temperature, for example 20° C., for a suitable period oftime, for example 6 hours. The product (IV) where Z is(3,4-dimethoxyphenyl)methanamine is isolated after separation from theby-products by filtration and purification.

Compounds of formula (IV) wherein Z is an OH group may be prepared byreaction of compounds of formula (XII):

wherein R₁ as hereinbefore defined for a compound of formula (I), with ahalogenating reagent, for example N-iodosuccinimide.

For example, a compound of formula (XII) is dissolved in a suitablesolvent, for example N,N-dimethylformamide, and reacted withN-iodosuccinimide at a suitable temperature, for example 60° C. for asuitable period of time, for example 1-2 hours. The product (XII) isisolated after an aqueous work-up and purification.

Compounds of formula (XII) may be prepared by reaction of compounds offormula (XIII):

wherein R₁ as hereinbefore defined for a compound of formula (I) with asuitable base, for example sodium hydroxide.

A solution of compounds of formula (XIII) in a suitable solvent, forexample ethyl alcohol, is treated with an aqueous solution of sodiumhydroxide and the reaction mixture stirred at a suitable temperature,for example 80-100° C. for a suitable period of time, for example 1-4hours. The product (XII) is isolated after an aqueous work-up andpurification.

Compounds of formula (XIII) can be prepared by reaction of compounds offormula (XIV):

with compounds of formula (XII):

wherein R₁ is as hereinbefore defined for a compound of formula (I).

For example a mixture of a compound of formula (XIV) and a compound offormula (XV) is treated with a solution of hydrogen chloride in asuitable solvent, for example a solution of hydrogen chloride in1,4-dioxane and is heated at a suitable temperature, 60-80° C. for asuitable period of time, for example 16-24 hours. The product (XIII) isisolated after evaporation of the solvent.

Compounds of formulae (VI), (VII), (XIV) and (XV) are either known inthe literature or are commercially available, for example fromSigma-Aldrich, UK, or may be prepared by analogy with known procedures,for example those disclosed in standard reference texts of syntheticmethodology such as J. March, Advanced Organic Chemistry, 6th Edition(2007), WileyBlackwell, or Comprehensive Organic Synthesis (Trost B. M.and Fleming I., (Eds.), Pergamon Press, 1991), each incorporated hereinby reference as it relates to such procedures.

Examples of other protecting groups that may be employed in thesynthetic routes described herein and the means for their removal can befound in T. W. Greene Protective Groups in Organic Synthesis; 4thEdition, J. Wiley and Sons, 2006, incorporated herein by reference as itrelates to such procedures.

For any of the hereinbefore described reactions or processes,conventional methods of heating and cooling may be employed, for exampletemperature-regulated oil-baths or temperature-regulated hot-blocks, andice/salt baths or dry ice/acetone baths respectively. Conventionalmethods of isolation, for example extraction from or into aqueous ornon-aqueous solvents may be used. Conventional methods of drying organicsolvents, solutions, or extracts, such as shaking with anhydrousmagnesium sulphate, or anhydrous sodium sulphate, or passing through ahydrophobic frit, may be employed. Conventional methods of purification,for example crystallisation and chromatography, for example silicachromatography or reverse-phase chromatography, may be used as required.Crystallisation may be performed using conventional solvents such asethyl acetate or methanol, ethanol, or butanol, and aqueous mixturesthereof. It will be appreciated that specific reaction timestemperatures may typically be determined by reaction-monitoringtechniques, for example thin-layer chromatography and LC-MS.

Where appropriate individual isomeric forms of the compounds of theinvention may be prepared as individual isomers using conventionalprocedures such as the fractional crystallisation of diastereoisomericderivatives or chiral high performance liquid chromatography (chiralHPLC).

The absolute stereochemistry of compounds may be determined usingconventional methods, such as X-ray crystallography.

Methods of Use

Examples of disease states in which the compounds of formula (I) andpharmaceutically acceptable salts thereof have potentially beneficialeffects include allergic diseases and other inflammatory conditions forexample allergic rhinitis and asthma, infectious diseases, and cancer.The compounds of formula (I) and pharmaceutically acceptable saltsthereof are also of potential use as vaccine adjuvants.

As modulators of the immune response the compounds of formula (I) andpharmaceutically acceptable salts thereof may also be useful in thetreatment and/or prevention of immune-mediated disorders, including butnot limited to inflammatory or allergic diseases such as asthma,allergic rhinitis and rhinoconjuctivitis, food allergy, hypersensitivitylung diseases, eosinophilic pneumonitis, delayed-type hypersensitivitydisorders, atherosclerosis, pancreatitis, gastritis, colitis,osteoarthritis, psoriasis, sarcoidosis, pulmonary fibrosis, respiratorydistress syndrome, bronchiolitis, chronic obstructive pulmonary disease,sinusitis, cystic fibrosis, actinic keratosis, skin dysplasia, chronicurticaria, eczema and all types of dermatitis.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be useful in the treatment and/or prevention ofreactions against respiratory infections, including but not limited toairways viral exacerbations and tonsillitis. The compounds may also beuseful in the treatment and/or prevention of autoimmune diseasesincluding but not limited to rheumatoid arthritis, psoriatic arthritis,systemic lupus erythematosus, Sjöegrens disease, ankylosing spondylitis,scleroderma, dermatomyositis, diabetes, graft rejection, includinggraft-versus-host disease, inflammatory bowel diseases including, butnot limited to, Crohn's disease and ulcerative colitis.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be useful in the treatment of infectious diseasesincluding, but not limited to, those caused by hepatitis viruses (e.g.hepatitis B virus, hepatitis C virus), human immunodeficiency virus,papillomaviruses, herpesviruses, respiratory viruses (e.g. influenzaviruses, respiratory syncytial virus, rhinovirus, metapneumovirus,parainfluenzavirus, SARS), and West Nile virus. The compounds of formula(I) and pharmaceutically acceptable salts thereof may also be useful inthe treatment of microbial infections caused by, for example, bacteria,fungi, or protozoa. These include, but are not limited to, tuberculosis,bacterial pneumonia, aspergillosis, histoplasmosis, candidosis,pneumocystosis, leprosy, chlamydia, cryptococcal disease,cryptosporidosis, toxoplasmosis, leishmania, malaria, andtrypanosomiasis.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be useful in the treatment of various cancers, inparticular the treatment of cancers that are known to be responsive toimmunotherapy and including, but not limited to, renal cell carcinoma,lung cancer, breast cancer, colorectal cancer, bladder cancer, melanoma,leukaemia, lymphomas and ovarian cancer.

It will be appreciated by those skilled in the art that referencesherein to treatment or therapy may, depending on the condition, extendto prophylaxis as well as the treatment of established conditions. Thereis thus provided as a further aspect of the invention a compound offormula (I), or a pharmaceutically acceptable salt thereof, for use intherapy.

It will be appreciated that, when a compound of formula (I) or apharmaceutically acceptable salt thereof is used in therapy, it is usedas an active therapeutic agent.

There is also therefore provided a compound of formula (I), or apharmaceutically acceptable salt thereof, for use in the treatment ofallergic diseases and other inflammatory conditions, infectious diseasesor cancer.

There is also therefore provided a compound of formula (I), or apharmaceutically acceptable salt thereof, for use in the treatment ofallergic rhinitis.

There is also therefore provided a compound of formula (I), or apharmaceutically acceptable salt thereof, for use in the treatment ofasthma.

There is further provided the use of a compound of formula (I), or apharmaceutically acceptable salt thereof, in the manufacture of amedicament for the treatment of allergic diseases and other inflammatoryconditions, infectious diseases or cancer.

There is further provided the use of a compound of formula (I), or apharmaceutically acceptable salt thereof, in the manufacture of amedicament for the treatment of allergic rhinitis.

There is further provided the use of a compound of formula (I), or apharmaceutically acceptable salt thereof, in the manufacture of amedicament for the treatment of asthma.

There is further provided a method of treatment of allergic diseases andother inflammatory conditions, infectious diseases or cancer, whichmethod comprises administering to a human subject in need thereof, atherapeutically effective amount of a compound of formula (I), or apharmaceutically acceptable salt thereof.

There is further provided a method of treatment of allergic rhinitis,which method comprises administering to a human subject in need thereof,a therapeutically effective amount of a compound of formula (I), or apharmaceutically acceptable salt thereof.

There is further provided a method of treatment of asthma, which methodcomprises administering to a human subject in need thereof, atherapeutically effective amount of a compound of formula (I), or apharmaceutically acceptable salt thereof.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof are also of potential use as vaccine adjuvants.

There is thus provided as a further aspect of the invention a vaccinecomposition comprising a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, and an antigen or antigen composition for usein therapy.

There is thus provided as a further aspect of the invention the use of acompound of formula (I), or a pharmaceutically acceptable salt thereof,and an antigen or antigen composition in the manufacture of a medicamentfor use in therapy.

There is further provided a method of treating or preventing diseasecomprising the administration to a human subject suffering from orsusceptible to disease, a vaccine composition comprising a compound offormula (I), or a pharmaceutically acceptable salt thereof, and anantigen or antigen composition.

There is further provided the use of a vaccine composition for themanufacture of a medicament for therapy.

Compositions

The compounds of formula (I) and pharmaceutically acceptable saltsthereof will normally, but not necessarily, be formulated intopharmaceutical compositions prior to administration to a patient.Accordingly, in another aspect of the invention there is provided apharmaceutical composition comprising a compound of formula (I), or apharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable excipients.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may be formulated for administration in any convenient way. Thecompounds of formula (I) and pharmaceutically acceptable salts thereofmay, for example, be formulated for oral, topical, inhaled, intranasal,buccal, parenteral (for example intravenous, subcutaneous, intradermal,or intramuscular) or rectal administration. In one aspect, the compoundsof formula (I) and pharmaceutically acceptable salts thereof areformulated for oral administration. In a further aspect, the compoundsof formula (I) and pharmaceutically acceptable salts thereof areformulated for topical administration, for example intranasal or inhaledadministration.

Tablets and capsules for oral administration may contain conventionalexcipients such as binding agents, for example syrup, acacia, gelatin,sorbitol, tragacanth, mucilage of starch, cellulose or polyvinylpyrrolidone; fillers, for example, lactose, microcrystalline cellulose,sugar, maize starch, calcium phosphate or sorbitol; lubricants, forexample, magnesium stearate, stearic acid, talc, polyethylene glycol orsilica; disintegrants, for example, potato starch, croscarmellose sodiumor sodium starch glycollate; or wetting agents such as sodium laurylsulphate. The tablets may be coated according to methods well known inthe art.

Oral liquid preparations may be in the form of, for example, aqueous oroily suspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may contain conventionaladditives such as suspending agents, for example, sorbitol syrup, methylcellulose, glucose/sugar syrup, gelatin, hydroxymethyl cellulose,carboxymethyl cellulose, aluminium stearate gel or hydrogenated ediblefats; emulsifying agents, for example, lecithin, sorbitan mono-oleate oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters, propyleneglycol or ethyl alcohol; or preservatives, for example, methyl or propylp-hydroxybenzoates or sorbic acid. The preparations may also containbuffer salts, flavouring, colouring and/or sweetening agents (e.g.mannitol) as appropriate.

Compositions for intranasal administration include aqueous compositionsadministered to the nose by drops or by pressurised pump. Suitablecompositions contain water as the diluent or carrier for this purpose.Compositions for administration to the lung or nose may contain one ormore excipients, for example one or more suspending agents, one or morepreservatives, one or more surfactants, one or more tonicity adjustingagents, one or more co-solvents, and may include components to controlthe pH of the composition, for example a buffer system. Further, thecompositions may contain other excipients such as antioxidants, forexample sodium metabisulphite, and taste-masking agents. Compositionsmay also be administered to the nose or other regions of the respiratorytract by nebulisation.

Intranasal compositions may permit the compound(s) of formula (I) or (a)pharmaceutically acceptable salt(s) thereof to be delivered to all areasof the nasal cavities (the target tissue) and further, may permit thecompound(s) of formula (I) or (a) pharmaceutically acceptable salt(s)thereof to remain in contact with the target tissue for longer periodsof time. A suitable dosing regime for intranasal compositions would befor the patient to inhale slowly through the nose subsequent to thenasal cavity being cleared. During inhalation the composition would beadministered to one nostril while the other is manually compressed. Thisprocedure would then be repeated for the other nostril. Typically, oneor two sprays per nostril would be administered by the above procedureone, two, or three times each day, ideally once daily. Of particularinterest are intranasal compositions suitable for once-dailyadministration.

The suspending agent(s), if included, will typically be present in anamount of from 0.1 to 5% (w/w), such as from 1.5% to 2.4% (w/w), basedon the total weight of the composition. Examples of pharmaceuticallyacceptable suspending agents include, but are not limited to, Avicel®(microcrystalline cellulose and carboxymethylcellulose sodium),carboxymethylcellulose sodium, veegum, tragacanth, bentonite,methylcellulose, xanthan gum, carbopol and polyethylene glycols.

Compositions for administration to the lung or nose may contain one ormore excipients may be protected from microbial or fungal contaminationand growth by inclusion of one or more preservatives. Examples ofpharmaceutically acceptable anti-microbial agents or preservativesinclude, but are not limited to, quaternary ammonium compounds (forexample benzalkonium chloride, benzethonium chloride, cetrimide,cetylpyridinium chloride, lauralkonium chloride and myristyl picoliniumchloride), mercurial agents (for example phenylmercuric nitrate,phenylmercuric acetate and thimerosal), alcoholic agents (for examplechlorobutanol, phenylethyl alcohol and benzyl alcohol), antibacterialesters (for example esters of para-hydroxybenzoic acid), chelatingagents such as disodium edetate (EDTA) and other anti-microbial agentssuch as chlorhexidine, chlorocresol, sorbic acid and its salts (such aspotassium sorbate) and polymyxin. Examples of pharmaceuticallyacceptable anti-fungal agents or preservatives include, but are notlimited to, sodium benzoate, sorbic acid, sodium propionate,methylparaben, ethylparaben, propylparaben and butylparaben. Thepreservative(s), if included, may be present in an amount of from 0.001to 1% (w/w), such as from 0.015% to 0.5% (w/w) based on the total weightof the composition.

Compositions (for example wherein at least one compound is insuspension) may include one or more surfactants which functions tofacilitate dissolution of the medicament particles in the aqueous phaseof the composition. For example, the amount of surfactant used is anamount which will not cause foaming during mixing. Examples ofpharmaceutically acceptable surfactants include fatty alcohols, estersand ethers, such as polyoxyethylene (20) sorbitan monooleate(Polysorbate 80), macrogol ethers, and poloxamers. The surfactant may bepresent in an amount of between about 0.01 to 10% (w/w), such as from0.01 to 0.75% (w/w), for example about 0.5% (w/w), based on the totalweight of the composition.

One or more tonicity-adjusting agent(s) may be included to achievetonicity with body fluids e.g. fluids of the nasal cavity, resulting inreduced levels of irritancy. Examples of pharmaceutically acceptabletonicity-adjusting agents include, but are not limited to, sodiumchloride, dextrose, xylitol, calcium chloride, glucose, glycerine andsorbitol. A tonicity-adjusting agent, if present, may be included in anamount of from 0.1 to 10% (w/w), such as from 4.5 to 5.5% (w/w), forexample about 5.0% (w/w), based on the total weight of the composition.

The compositions of the invention may be buffered by the addition ofsuitable buffering agents such as sodium citrate, citric acid,trometamol, phosphates such as disodium phosphate (for example thedodecahydrate, heptahydrate, dihydrate and anhydrous forms), or sodiumphosphate and mixtures thereof.

A buffering agent, if present, may be included in an amount of from 0.1to 5% (w/w), for example 1 to 3% (w/w) based on the total weight of thecomposition.

Examples of taste-masking agents include sucralose, sucrose, saccharinor a salt thereof, fructose, dextrose, glycerol, corn syrup, aspartame,acesulfame-K, xylitol, sorbitol, erythritol, ammonium glycyrrhizinate,thaumatin, neotame, mannitol, menthol, eucalyptus oil, camphor, anatural flavouring agent, an artificial flavouring agent, andcombinations thereof.

One or more co-solvent(s) may be included to aid solubility of themedicament compound(s) and/or other excipients. Examples ofpharmaceutically acceptable co-solvents include, but are not limited to,propylene glycol, dipropylene glycol, ethylene glycol, glycerol,ethanol, polyethylene glycols (for example PEG300 or PEG400), andmethanol. In one embodiment, the co-solvent is propylene glycol.

Co-solvent(s), if present, may be included in an amount of from 0.05 to30% (w/w), such as from 1 to 25% (w/w), for example from 1 to 10% (w/w)based on the total weight of the composition.

Compositions for inhaled administration include aqueous, organic oraqueous/organic mixtures, dry powder or crystalline compositionsadministered to the respiratory tract by pressurised pump or inhaler,for example, reservoir dry powder inhalers, unit-dose dry powderinhalers, pre-metered multi-dose dry powder inhalers, nasal inhalers orpressurised aerosol inhalers, nebulisers or insufflators. Suitablecompositions contain water as the diluent or carrier for this purposeand may be provided with conventional excipients such as bufferingagents, tonicity modifying agents and the like. Aqueous compositions mayalso be administered to the nose and other regions of the respiratorytract by nebulisation. Such compositions may be aqueous solutions orsuspensions or aerosols delivered from pressurised packs, such as ametered dose inhaler, with the use of a suitable liquefied propellant.

Compositions for administration topically to the nose (for example, forthe treatment of rhinitis) or to the lung, include pressurised aerosolcompositions and aqueous compositions delivered to the nasal cavities bypressurised pump. Compositions which are non-pressurised and aresuitable for administration topically to the nasal cavity are ofparticular interest. Suitable compositions contain water as the diluentor carrier for this purpose. Aqueous compositions for administration tothe lung or nose may be provided with conventional excipients such asbuffering agents, tonicity-modifying agents and the like. Aqueouscompositions may also be administered to the nose by nebulisation.

A fluid dispenser may typically be used to deliver a fluid compositionto the nasal cavities. The fluid composition may be aqueous ornon-aqueous, but typically aqueous. The compound of formula (I), or apharmaceutically acceptable salt thereof, may be formulated as asuspension or solution. Such a fluid dispenser may have a dispensingnozzle or dispensing orifice through which a metered dose of the fluidcomposition is dispensed upon the application of a user-applied force toa pump mechanism of the fluid dispenser. Such fluid dispensers aregenerally provided with a reservoir of multiple metered doses of thefluid composition, the doses being dispensable upon sequential pumpactuations. Alternatively, the fluid dispenser for delivery of a fluidcomposition to the nasal cavities may be designed to be dose-limited,for example a single use dispenser comprising a single dose. Thedispensing nozzle or orifice may be configured for insertion into thenostrils of the user for spray dispensing of the fluid composition intothe nasal cavity. A fluid dispenser of the aforementioned type isdescribed and illustrated in International Patent Applicationpublication number WO 2005/044354 (Glaxo Group Limited). The dispenserhas a housing which houses a fluid-discharge device having a compressionpump mounted on a container for containing a fluid composition. Thehousing has at least one finger-operable side lever which is movableinwardly with respect to the housing to move the container upwardly inthe housing by means of a cam to cause the pump to compress and pump ametered dose of the composition out of a pump stem through a nasalnozzle of the housing. In one embodiment, the fluid dispenser is of thegeneral type illustrated in FIGS. 30-40 of WO 2005/044354.

Aqueous compositions containing a compound of formula (I) or apharmaceutically acceptable salt thereof may also be delivered by a pumpas disclosed in International Patent Application publication numberWO2007/138084 (Glaxo Group Limited), for example as disclosed withreference to FIGS. 22-46 thereof, or as disclosed in United Kingdompatent application number GB0723418.0 (Glaxo Group Limited), for exampleas disclosed with reference to FIGS. 7-32 thereof. The pump may beactuated by an actuator as disclosed in FIGS. 1-6 of GB0723418.0.

Dry powder compositions for topical delivery to the lung by inhalationmay, for example, be presented in capsules and cartridges of for examplegelatine, or blisters of for example laminated aluminium foil, for usein an inhaler or insufflator. Powder blend compositions generallycontain a powder mix for inhalation of the compound of formula (I) or apharmaceutically acceptable salt thereof and a suitable powder base(carrier/diluent/excipient substance) such as mono-, di-, orpolysaccharides (for example lactose or starch). Dry powder compositionsmay also include, in addition to the drug and carrier, a furtherexcipient (for example a ternary agent such as a sugar ester for examplecellobiose octaacetate, calcium stearate, or magnesium stearate.

In one embodiment, a composition suitable for inhaled administration maybe incorporated into a plurality of sealed dose containers provided onmedicament pack(s) mounted inside a suitable inhalation device. Thecontainers may be rupturable, peelable, or otherwise openableone-at-a-time and the doses of the dry powder composition administeredby inhalation on a mouthpiece of the inhalation device, as known in theart.

The medicament pack may take a number of different forms, for instance adisk-shape or an elongate strip. Representative inhalation devices arethe DISKHALER™ and DISKUS™ devices, marketed by GlaxoSmithKline.

A dry powder inhalable composition may also be provided as a bulkreservoir in an inhalation device, the device then being provided with ametering mechanism for metering a dose of the composition from thereservoir to an inhalation channel where the metered dose is able to beinhaled by a patient inhaling at a mouthpiece of the device. Exemplarymarketed devices of this type are TURBUHALER™ (AstraZeneca), TWISTHALER™(Schering) and CLICKHALER™ (Innovata.) A further delivery method for adry powder inhalable composition is for metered doses of the compositionto be provided in capsules (one dose per capsule) which are then loadedinto an inhalation device, typically by the patient on demand. Thedevice has means to rupture, pierce or otherwise open the capsule sothat the dose is able to be entrained into the patient's lung when theyinhale at the device mouthpiece. As marketed examples of such devicesthere may be mentioned ROTAHALER™ (GlaxoSmithKline) and HANDIHALER™(Boehringer Ingelheim.)

Pressurised aerosol compositions suitable for inhalation can be either asuspension or a solution and may contain a compound of formula (I) or apharmaceutically acceptable salt thereof and a suitable propellant suchas a fluorocarbon or hydrogen-containing chlorofluorocarbon or mixturesthereof, particularly hydrofluoroalkanes, especially1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoro-n-propane or amixture thereof. The aerosol composition may optionally containadditional composition excipients well known in the art such assurfactants e.g. oleic acid, lecithin or an oligolactic acid orderivative thereof e.g. as described in WO 94/21229 and WO 98/34596(Minnesota Mining and Manufacturing Company) and co-solvents e.g.ethanol. Pressurised compositions will generally be retained in acanister (e.g. an aluminium canister) closed with a valve (e.g. ametering valve) and fitted into an actuator provided with a mouthpiece.

Ointments, creams and gels, may, for example, be formulated with anaqueous or oily base with the addition of suitable thickening and/orgelling agent and/or solvents. Such bases may thus, for example, includewater and/or an oil such as liquid paraffin or a vegetable oil such asarachis oil or castor oil, or a solvent such as polyethylene glycol.Thickening agents and gelling agents which may be used according to thenature of the base include soft paraffin, aluminium stearate,cetostearyl alcohol, polyethylene glycols, wool-fat, beeswax,carboxypolymethylene and cellulose derivatives, and/or glycerylmonostearate and/or non-ionic emulsifying agents.

Lotions may be formulated with an aqueous or oily base and will ingeneral also contain one or more emulsifying agents, stabilising agents,dispersing agents, suspending agents or thickening agents.

Powders for external application may be formed with the aid of anysuitable powder base, for example, talc, lactose or starch. Drops may beformulated with an aqueous or non-aqueous base also comprising one ormore dispersing agents, solubilising agents, suspending agents orpreservatives.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may, for example, be formulated for transdermal delivery bycomposition into patches or other devices (e.g. pressurised gas devices)which deliver the active component into the skin.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in the conventional manner.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be formulated as suppositories, e.g. containingconventional suppository bases such as cocoa butter or other glycerides.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be formulated for parenteral administration by bolusinjection or continuous infusion and may be presented in unit dose form,for instance as ampoules, vials, small volume infusions or pre-filledsyringes, or in multidose containers with an added preservative. Thecompositions may take such forms as solutions, suspensions, or emulsionsin aqueous or non-aqueous vehicles, and may contain formulatory agentssuch as antioxidants, buffers, antimicrobial agents and/or tonicityadjusting agents. Alternatively, the active ingredient may be in powderform for constitution with a suitable vehicle, e.g. sterile,pyrogen-free water, before use. The dry solid presentation may beprepared by filling a sterile powder aseptically into individual sterilecontainers or by filling a sterile solution aseptically into eachcontainer and freeze-drying.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be formulated with vaccines as adjuvants to modulatetheir activity. Such compositions may contain antibody(ies) or antibodyfragment(s) or an antigenic component including but not limited toprotein, DNA, live or dead bacteria and/or viruses or virus-likeparticles, together with one or more components with adjuvant activityincluding but not limited to aluminium salts, oil and water emulsions,heat shock proteins, lipid A preparations and derivatives, glycolipids,other TLR agonists such as CpG DNA or similar agents, cytokines such asGM-CSF or IL-12 or similar agents.

In a further aspect of the invention, there is provided a vaccineadjuvant comprising a compound of formula (I), or a pharmaceuticallyacceptable salt thereof. There is further provided a vaccine compositioncomprising a compound of formula (I), or a pharmaceutically acceptablesalt thereof, and an antigen or antigen composition.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may be employed alone or in combination with othertherapeutically-active agents. The invention provides in a furtheraspect, a combination comprising a compound of formula (I), or apharmaceutically acceptable salt thereof, together with at least oneother therapeutically-active agent.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof and the other therapeutically-active agent(s) may beadministered together or separately and, when administered separately,administration may occur simultaneously or sequentially, in any order.The amounts of the compound(s) of formula (I) or (a) pharmaceuticallyacceptable salt(s) thereof and the other therapeutically-active agent(s)and the relative timings of administration will be selected in order toachieve the desired combined therapeutic effect. The administration of acombination of a compound of formula (I) or a pharmaceuticallyacceptable salt thereof with other treatment agents may be byadministration concomitantly in a unitary pharmaceutical compositionincluding both compounds, or in separate pharmaceutical compositionseach including one of the compounds. Alternatively, the combination maybe administered separately in a sequential manner wherein one treatmentagent is administered first and the other second or vice versa. Suchsequential administration may be close in time or remote in time.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may be used in combination with one or more agents useful in theprevention or treatment of viral infections. Examples of such agentsinclude, without limitation; polymerase inhibitors such as thosedisclosed in WO 2004/037818-A1, as well as those disclosed in WO2004/037818 and WO 2006/045613; JTK-003, JTK-019, NM-283, HCV-796,R-803, R1728, R1626, as well as those disclosed in WO 2006/018725, WO2004/074270, WO 2003/095441, US2005/0176701, WO 2006/020082, WO2005/080388, WO 2004/064925, WO 2004/065367, WO 2003/007945, WO02/04425, WO 2005/014543, WO 2003/000254, EP 1065213, WO 01/47883, WO2002/057287, WO 2002/057245 and similar agents; replication inhibitorssuch as acyclovir, famciclovir, ganciclovir, cidofovir, lamivudine andsimilar agents; protease inhibitors such as the HIV protease inhibitorssaquinavir, ritonavir, indinavir, nelfinavir, amprenavir, fosamprenavir,brecanavir, atazanavir, tipranavir, palinavir, lasinavir, and the HCVprotease inhibitors BILN2061, VX-950, SCHSO3034; and similar agents;nucleoside and nucleotide reverse transcriptase inhibitors such aszidovudine, didanosine, lamivudine, zalcitabine, abacavir, stavidine,adefovir, adefovir dipivoxil, fozivudine, todoxil, emtricitabine,alovudine, amdoxovir, elvucitabine, and similar agents; non-nucleosidereverse transcriptase inhibitors (including an agent havinganti-oxidation activity such as immunocal, oltipraz etc.) such asnevirapine, delavirdine, efavirenz, loviride, immunocal, oltipraz,capravirine, TMC-278, TMC-125, etravirine, and similar agents; entryinhibitors such as enfuvirtide (T-20), T-1249, PRO-542, PRO-140,TNX-355, BMS-806, 5-Helix and similar agents; integrase inhibitors suchas L-870,180 and similar agents; budding inhibitors such as PA-344 andPA-457, and similar agents; chemokine receptor inhibitors such asvicriviroc (Sch-C), Sch-D, TAK779, maraviroc (UK-427,857), TAK449, aswell as those disclosed in WO 02/74769, WO 2004/054974, WO 2004/055012,WO 2004/055010, WO 2004/055016, WO 2004/055011, and WO 2004/054581, andsimilar agents; neuraminidase inhibitors such as CS-8958, zanamivir,oseltamivir, peramivir and similar agents; ion channel blockers such asamantadine or rimantadine and similar agents; and interfering RNA andantisense oligonucleotides and such as ISIS-14803 and similar agents;antiviral agents of undetermined mechanism of action, for example thosedisclosed in WO 2005/105761, WO 2003/085375, WO 2006/122011, ribavirin,and similar agents. The compounds of formula (I) and pharmaceuticallyacceptable salts thereof may also be used in combination with one ormore other agents which may be useful in the prevention or treatment ofviral infections for example immune therapies (e.g. interferon or othercytokines/chemokines, cytokine/chemokine receptor modulators, cytokineagonists or antagonists and similar agents); and therapeutic vaccines,antifibrotic agents, anti-inflammatory agents such as corticosteroids orNSAIDs (non-steroidal anti-inflammatory agents) and similar agents.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may be used in combination with one or more other agents whichmay be useful in the prevention or treatment of allergic disease,inflammatory disease, autoimmune disease, for example; antigenimmunotherapy, anti-histamines, steroids, NSAIDs, bronchodilators (e.g.beta 2 agonists, adrenergic agonists, anticholinergic agents,theophylline), methotrexate, leukotriene modulators and similar agents;monoclonal antibody therapy such as anti-IgE, anti-TNF, anti-IL-5,anti-IL-6, anti-IL-12, anti-IL-1 and similar agents; receptor therapiese.g. entanercept and similar agents; antigen non-specificimmunotherapies (e.g. interferon or other cytokines/chemokines,cytokine/chemokine receptor modulators, cytokine agonists orantagonists, TLR agonists and similar agents).

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may be used in combination with one or more other agents whichmay be useful in the prevention or treatment of cancer, for examplechemotherapeutics such as alkylating agents, topoisomerase inhibitors,antimetabolites, antimitotic agents, kinase inhibitors and similaragents; monoclonal antibody therapy such as trastuzumab, gemtuzumab andother similar agents; and hormone therapy such as tamoxifen, goserelinand similar agents.

The pharmaceutical compositions according to the invention may also beused alone or in combination with at least one other therapeutic agentin other therapeutic areas, for example gastrointestinal disease. Thecompositions according to the invention may also be used in combinationwith gene replacement therapy.

The invention includes in a further aspect a combination comprising acompound of formula (I), or a pharmaceutically acceptable salt thereof,together with at least one other therapeutically active agent.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical composition and thus pharmaceuticalcompositions comprising a combination as defined above together with atleast one pharmaceutically acceptable diluent or carrier thereofrepresent a further aspect of the invention.

A therapeutically effective amount of a compound of formula (I) or apharmaceutically acceptable salt thereof will depend upon a number offactors. For example, the species, age, and weight of the recipient, theprecise condition requiring treatment and its severity, the nature ofthe composition, and the route of administration are all factors to beconsidered. The therapeutically effective amount ultimately should be atthe discretion of the attendant physician. Regardless, an effectiveamount of a compound of the present invention for the treatment ofhumans suffering from frailty, generally, should be in the range of0.0001 to 100 mg/kg body weight of recipient per day. More usually theeffective amount should be in the range of 0.001 to 10 mg/kg body weightper day. Thus, for a 70 kg adult one example of an actual amount per daywould usually be from 7 to 700 mg. For intranasal and inhaled routes ofadministration, typical doses for a 70 kg adult should be in the rangeof 0.1 micrograms to 1 mg per day, for example 1 μg, 10 μg or 100 μg.This amount may be given in a single dose per day or in a number (suchas two, three, four, five, or more) of sub-doses per day such that thetotal daily dose is the same. An effective amount of a pharmaceuticallyacceptable salt of a compound of formula (I) may be determined as aproportion of the effective amount of the compound of formula (I) or apharmaceutically acceptable salt thereof per se. Similar dosages shouldbe appropriate for treatment of the other conditions referred to herein.

Compounds of formula (I) and pharmaceutically acceptable salts thereofmay also be administered at any appropriate frequency e.g. 1-7 times perweek. The precise dosing regimen will of course depend on factors suchas the therapeutic indication, the age and condition of the patient, andthe particular route of administration chosen. In one aspect of theinvention, a compound of formula (I), or a pharmaceutically acceptablesalt thereof, may be administered once weekly for a period of 4 to 8weeks, for example 4, 5, 6, 7 or 8 weeks. Repeat treatment cycles may berequired.

Pharmaceutical compositions may be presented in unit-dose formscontaining a predetermined amount of active ingredient per unit dose.Such a unit may contain, as a non-limiting example, 0.5 mg to 1 g of acompound of formula (I) or a pharmaceutically acceptable salt thereof,depending on the condition being treated, the route of administration,and the age, weight, and condition of the patient. Preferred unit-dosagecompositions are those containing a daily dose or sub-dose, as hereinabove recited, or an appropriate fraction thereof, of an activeingredient. Such pharmaceutical compositions may be prepared by any ofthe methods well-known in the pharmacy art.

There is also provided a process for preparing such a pharmaceuticalcomposition which comprises admixing a compound of formula (I), or apharmaceutically acceptable salt thereof, with one or morepharmaceutically acceptable excipients.

ABBREVIATIONS

The following list provides definitions of certain abbreviations as usedherein. It will be appreciated that the list is not exhaustive, but themeaning of those abbreviations not herein below defined will be readilyapparent to those skilled in the art.

-   DCM Dichloromethane-   DMF N,N-Dimethylformamide-   DMSO Dimethylsulphoxide-   DME 1,2-Dimethoxyethane-   THF Tetrahydrofuran-   EtOAc Ethyl acetate-   MeOH Methanol-   EtOH Ethanol-   MeCN Acetonitrile-   HCl Hydrochloric acid-   HPLC High performance liquid chromatography-   IPA iso-Propanol-   MDAP Mass Directed Autopreparative HPLC-   SPE Solid phase extraction-   MeOH Methanol-   TFA Trifluoroacetic acid-   DIPEA N,N-Diisopropylethylamine

Experimental Details

NMR

¹H NMR spectra were recorded in either CDCl₃ or DMSO-d₆ on either aBruker DPX 400 or Bruker Avance DRX, Varian Unity 400 spectrometer orJEOL Delta all working at 400 MHz. The internal standard used was eithertetramethylsilane or the residual protonated solvent at 7.25 ppm forCDCl₃ or 2.50 ppm for DMSO-d₆.

LCMS

System A

-   Column: 50 mm×2.1 mm ID, 1.7 μm Acquity UPLC BEH C₁₈-   Flow Rate: 1 mL/min.-   Temp: 40° C.-   UV detection range: 210 to 350 nm-   Mass spectrum: Recorded on a mass spectrometer using    alternative-scan positive and-   negative mode electrospray ionisation-   Solvents: A: 0.1% v/v formic acid in water    -   B: 0.1% v/v formic acid acetonitrile

Gradient: Time (min.) A % B % 0 97 3 1.5 0 100 1.9 0 100 2.0 97 3System B

-   Column: 50 mm×2.1 mm ID, 1.7 μm Acquity UPLC BEH C₁₈-   Flow Rate: 1 mL/min.-   Temp: 40° C.-   UV detection range: 210 to 350 nm-   Mass spectrum: Recorded on a mass spectrometer using    alternative-scan positive and negative mode electrospray ionisation-   Solvents: A: 10 mM ammonium bicarbonate in water adjusted to pH10    with ammonia solution    -   B: acetonitrile

Gradient: Time (min.) A % B % 0 99 1 1.5 3 97 1.9 3 97 2.0 0 100Mass Directed Autopreoarative HPLC (MDAP)

Mass directed autopreparative HPLC was undertaken under the conditionsgiven below. The UV detection was an averaged signal from wavelength of210 nm to 350 nm and mass spectra were recorded on a mass spectrometerusing alternate-scan positive and negative mode electrospray ionization.

Method A

Method A was conducted on a Sunfire C₁₈ column (typically 150 mm×30 mmi.d. 5 μm packing diameter) at ambient temperature. The solventsemployed were:

-   A=0.1% v/v solution of formic acid in water-   B=0.1% v/v solution of formic acid in acetonitrile.    Method B

Method B was conducted on an XBridge C₁₈ column (typically 100 mm×30 mmi.d. 5 μm packing diameter) at ambient temperature. The solventsemployed were:

A=10 mM aqueous ammonium bicarbonate adjusted to pH 10 with ammoniasolution. B=acetonitrile.

INTERMEDIATE PREPARATION Intermediate 15-Butyl-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

A 4M solution of hydrogen chloride in 1,4-dioxane (19.4 mL, 78 mmol) wasadded to a suspension of ethyl 4-amino-1H-pyrazole-5-carboxylatehydrochloride (2 g, 10.44 mmol) in valeronitrile (94 mL) at roomtemperature. The resultant mixture was stirred at 80° C. for 22 hours.The cooled reaction mixture was evaporated in-vacuo to yield a brownsolid which was dissolved in ethanol (29 mL), added to a solution ofsodium hydroxide (1.67 g, 41.7 mmol) in water (7.1 mL) and stirred at100° C. for 1 hour. The reaction was cooled to room temperature, dilutedwith water (65 mL) and the pH adjusted to 10 using 2M aqueous citricacid solution. The reaction mixture was extracted with ethyl acetate(220 mL). The organic phase was separated, passed through a hydrophobicfrit and evaporated in-vacuo to yield a brown solid. The retainedaqueous was adjusted to pH 7 using 2M aqueous citric acid solution andextracted with ethyl acetate (220 mL). The organic phase was separated,passed through a hydrophobic frit and evaporated in-vacuo to yield abrown solid. The two batches of brown solid were combined to yield asolid (3.3 g). N-Iodosuccinimide (3.52 g, 15.66 mmol) was addedportionwise to a solution of the solid (3.3 g) in anhydrousN,N-dimethylformamide (43 mL) at room temperature. The mixture wasstirred at 60° C. for 2 hours. The cooled reaction was evaporatedin-vacuo and partitioned between ethyl acetate and water/brine (1:1).The organic layer was separated, passed through a hydrophobic frit andevaporated in-vacuo to yield a brown solid (6.3 g). The solid wasdissolved in ethyl acetate, loaded onto 2×50 g ISOLUTE NH₂ cartridgesand purified using ethyl acetate (2×400 mL), 5% methanol indichloromethane (2×200 mL), 10% methanol in dichloromethane (2×100 mL),15% methanol in dichloromethane (2×100 mL) and finally 20% methanol indichloromethane (2×700 mL) as eluent. Appropriate fractions werecombined and evaporated in-vacuo to yield the title compound as a whitesolid (2.12 g).

LCMS (System A): t_(RET)=0.79 min; MH⁺319

Intermediate 2 1-(Hex-5-r-1-yl)piperidine

A solution of 6-chlorohex-1-yne (5 mL, 41.3 mmol), piperidine (4.08 mL,41.3 mmol) and sodium hydrogen carbonate (4.16 g, 49.5 mmol) in DMF (50mL) was refluxed for 16 hours. The reaction was concentrated in-vacuoand the residue partitioned between ether (150 mL) and water (150 mL).The organic was separated and the aqueous back extracted with diethylether (50 mL). The combined organics were washed with brine (150 mL),dried (MgSO₄), filtered and concentrated in vacuo to give a crude sampleof the title compound (3.74 g). Oxalic acid (2.161 g, 24 mmol) was addedto the crude product. The resultant solid was recrystallised fromethanol, collected by filtration and dried in-vacuo to give1-(hex-5-yn-1-yl)piperidine oxalic acid salt (4.66 g). The solid waspartitioned between diethyl ether (150 mL) and saturated aqueous sodiumbicarbonate (150 mL). The organic was separated and dried (MgSO₄)filtered and concentrated in vacuo to give the title compound as ayellow oil (1.93 g).

¹H NMR (400 MHz, CHLOROFORM-d) δppm 2.31-2.52 (m, 6 H) 2.18-2.26 (m, 2H) 1.92-1.96 (m, 1 H) 1.40-1.72 (m, 10 H)

Intermediate 35-Butyl-3-(6-(piperidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

To a degassed solution of5-butyl-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (300 mg, 0.943mmol) in anhydrous N,N-dimethylformamide (7 mL) under nitrogenatmosphere at room temperature was added copper(I) iodide (36 mg, 0.189mmol), tetrakis(triphenylphosphine)-palladium(0) (120 mg, 0.104 mmol)and finally triethylamine (0.289 mL, 2.075 mmol). The mixture wasstirred at room temperature under nitrogen atmosphere for 10 minutes andthen a solution of 1-(5-hexyn-1-yl)piperidine (343 mg, 2.075 mmol) inanhydrous N,N-dimethylformamide (1 mL) was added. The reaction mixturewas stirred at room temperature for 23 hours. The reaction wasevaporated in-vacuo to yield a brown oil, the oil was diluted withdichloromethane (15 mL), loaded onto a 70 g ISOLUTE NH₂ cartridge andpurified by chromatography using a 0-25% methanol in dichloromethanegradient over 80 minutes (UV collection wavelength set to 233 nm).Appropriate fractions were combined and evaporated in-vacuo to yield apale yellow oil (330 mg). A solution of the oil (330 mg) in ethanol (50mL) was passed through the H-cube (settings: 45° C., full hydrogen, 1mL/min flow rate and 10% palladium on carbon CatCart30 as the catalyst).The solution was evaporated in-vacuo to yield a colourless oil. The oilwas dissolved in MeOH:DMSO (1:1) (4×1 mL) and purified by MDAP (MethodB). Appropriate fractions were combined and evaporated in-vacuo to yielda white solid (205 mg) A solution of the solid (205 mg) in ethanol (40mL) was passed through the H-cube (settings: 45° C., full hydrogen, 1mL/min flow rate and 10% palladium on carbon CatCart30 as the catalyst).The solution was evaporated in-vacuo to yield the title compound a whitesolid (201 mg).

LCMS (System B): t_(RET)=0.93 min; MH⁺360

Intermediate 43-Iodo-5-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

A 4M solution of hydrogen chloride in 1,4-dioxane (5.19 mL, 20.74 mmol)was added to a suspension of ethyl 4-amino-1H-pyrazole-5-carboxylatehydrochloride (535 mg, 2.79 mmol) in 3-methoxypropanenitrile (25.7 mL,240 mmol) at room temperature. The resultant mixture was stirred at 80°C. for 2.5 hours. The cooled reaction mixture was evaporated in-vacuo toyield a pale yellow solid which was dissolved in ethanol (7.7 mL), addedto a solution of sodium hydroxide (447 mg, 11.17 mmol) in water (1.9 mL)and stirred at room temperature for 20 minutes. The reaction wasevaporated in-vacuo to yield a brown solid. N-Iodosuccinimide (942 mg,4.19 mmol) was added portionwise to a solution of the above compound inanhydrous N,N-dimethylformamide (DMF) (11.3 mL) at room temperature. Themixture was stirred at 60° C. for 45 minutes. The cooled reaction wasevaporated in-vacuo.

The residue was dissolved in the minimum volume of dichloromethane,loaded onto a 2×50 g ISOLUTE NH₂ cartridge and purified bychromatography using a 0-50% MeOH/DCM gradient over 60 minutes.Appropriate fractions were combined and evaporated in-vacuo to yield acream solid (203 mg). Impure fractions from the column containingdesired product were combined, concentrated in vacuo then repurified bychromatography using the same method as above. The products from bothcolumns were combined to give the title compound as a yellow solid (309mg).

LCMS (System A): t_(RET)=0.54 min; MH⁺321

Intermediate 55-(2-Methoxyethyl)-3-(6-(piperidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

To a degassed solution of3-iodo-5-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (232 mg,0.724 mmol) in anhydrous N,N-dimethylformamide (5 mL) under a nitrogenatmosphere at room temperature was added copper(I) iodide (27.6 mg,0.145 mmol), tetrakis(triphenylphosphine)-palladium(0) (92 mg, 0.080mmol) and finally triethylamine (0.222 mL, 1.592 mmol). The mixture wasstirred at room temperature under nitrogen atmosphere for 10 minutes andthen a solution of 1-(5-hexyn-1-yl)piperidine (263 mg, 1.592 mmol) inanhydrous N,N-dimethylformamide (1 mL) was added. The reaction mixturewas stirred at room temperature for 20 hours then heated to 55° C. andleft to stir for 5 hours. The reaction was evaporated in-vacuo to yielda brown oil. The oil was diluted with dichloromethane, loaded onto a 50g ISOLUTE NH₂ cartridge and purified by chromatography using a 0-50%methanol in dichloromethane gradient over 60 minutes (UV collectionwavelength set to 230 nm). Appropriate fractions were combined andevaporated in-vacuo to yield a pale yellow oil (188 mg). A solution ofthe oil (188 mg) in ethanol (30 mL) was passed through the H-cube(settings: 45° C., full hydrogen, 1 mL/min flow rate and 10% palladiumon carbon CatCart30 as the catalyst). The solution was evaporatedin-vacuo to yield a pale yellow oil and the crude product was purifiedby MDAP (Method B). Appropriate fractions were combined and evaporatedin-vacuo to yield the title compound as a clear oil (117 mg).

LCMS (System B): t_(RET)=0.76 min; MH⁺362

Intermediate 65-Butyl-3-(6-chlorohex-1-yn-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

To a solution of 5-butyl-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one(4.97 g, 15.62 mmol) in N,N-dimethylformamide (100 mL) was addedbis(triphenylphosphine)palladium(II)dichloride (1.228 g, 1.750 mmol) andcopper(I) iodide (0.595 g, 3.12 mmol). The solution was stirred anddegassed with nitrogen for 5 minutes then the reaction mixture placedunder a nitrogen atmosphere. A solution of 6-chloro-1-hexyne (3.64 g,31.2 mmol) and triethylamine (4.36 mL, 31.2 mmol) inN,N-dimethylformamide (30 mL) was added dropwise over 10 minutes. Thereaction mixture was stirred at ambient temperature for a further 10minutes then heated to 60° C. for 2.5 hours. The reaction mixture wasconcentrated in vacuo at 60° C. and the resulting residue partitionedbetween ethyl acetate (250 mL) and a 1:1 mixture of water:brine (500mL). The organic layer was separated and the aqueous layer backextracted with ethyl acetate (250 mL). The combined organic phases weredried through a hydrophobic frit and concentrated in vacuo to give abrown solid (8.0 g). The residue was dissolved in a 1:1 mixture ofMeOH:DCM and absorbed onto Florisil. The solid was loaded and purifiedby chromatography on silica (330 g) using a 0-100% ethylacetate-cyclohexane gradient over 10 column volumes followed by flushingwith ethyl acetate for 9 column volumes. The appropriate fractions werecombined and evaporated in vacuo to give a yellow solid (3.55 g). Thesolid was triturated with diisopropyl ether, filtered and dried in vacuoat 50° C. to give the title compound as a pale yellow solid (3.14 g).

LCMS (System B): t_(RET)=0.98 min; MH⁺307, 309

Intermediate 75-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

To a suspension of5-butyl-3-(6-chlorohex-1-yn-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one(2.59 g, 8.44 mmol) in anhydrous acetonitrile (115 mL) was addedpyrrolidine (2.11 mL, 25.3 mmol) and triethylamine (3.53 mL, 25.3 mmol).The reaction was stirred at 80° C. for 3.5 hours. A further 2.11 mL(25.3 mmol) of pyrrolidine and 3.53 mL (25.3 mmol) of triethylamine wereadded to the reaction. The reaction was stirred at 80° C. for a further18 hours. The cooled reaction was partitioned between ethyl acetate andwater. The organic layer was separated, passed through a hydrophobicfrit and evaporated in-vacuo to yield a yellow oil (1.9 g). The retainedaqueous was re-extracted with 20% methanol in dichloromethane. Theorganic layer was passed through a hydrophobic frit and evaporatedin-vacuo to yield a yellow oil (1.02 g). The two yellow oils werecombined to yield a yellow oil (2.92 g). A mixture of the oil and 10 wt% palladium on carbon (350 mg) in ethanol (120 mL) was stirred under ahydrogen atmosphere at room temperature for 90 minutes. A further 350 mgof 10 wt % palladium on carbon was added to the reaction under anitrogen atmosphere and the reaction was stirred under a hydrogenatmosphere at room temperature for 60 minutes. A further 350 mg of 10 wt% palladium on carbon was added to the reaction under a nitrogenatmosphere and the reaction was stirred under a hydrogen atmosphere atroom temperature for 60 minutes. The reaction mixture was filteredthrough a 10 g celite cartridge and the filtrate evaporated in-vacuo toyield a yellow oil (2.8 g). The oil was dissolved in the minimum volumeof dichloromethane, loaded onto a 375 g Biotage KP-NH cartridge andpurified using a 0-10% methanol in dichloromethane gradient over 12column volumes followed by 10% methanol in dichloromethane over 3 columnvolumes. Appropriate fractions were combined and evaporated in-vacuo toyield the title compound as a pale yellow solid (1.845 g).

LCMS (System B): t_(RET)=0.85 min; MH⁺346

Impure fractions from the chromatography were combined and evaporatedin-vacuo to yield a yellow oil (380 mg). The oil was dissolved inMeOH:DMSO (1:1) (4×1 ml) and purified by MDAP (Method B). Appropriatefractions were combined and evaporated in-vacuo to yield a furtherportion of the title compound a pale yellow oil (198 mg).

Intermediate 85-Butyl-N-(3,4-dimethoxybenzyl)-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-amine

To a solution of 5-butyl-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (5g, 15.72 mmol) and (benzotriazol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate (10.43 g, 23.58 mmol) in anhydrous DMF (250 mL) atroom temperature under a nitrogen atmosphere was added1,8-diazabicyclo[5.4.0]undec-7-ene (4.23 mL, 28.3 mmol) and the reactionmixture was stirred for 2.5 hours. (3,4-Dimethoxyphenyl)methanamine (20mL, 94 mmol) was added and the mixture was warmed to 40° C. for 3 hours.The reaction mixture was evaporated in-vacuo and the resulting oilpartitioned between ethyl acetate and water. The organic layer wasseparated, washed with brine, dried over MgSO₄, filtered and evaporatedin-vacuo to yield as a semi-crystalline yellow oil (23.7 g). The residuewas absorbed onto Florosil, loaded onto a 330 g silica pre-conditionedcartridge and purified by chromatography using cyclohexane over 1 columnvolume followed by 0-100% ethyl acetate in cyclohexane gradient over 14column volumes followed by ethyl acetate over 4 column volumes.Appropriate fractions containing only the desired material by LC-MSanalysis were combined and evaporated in-vacuo to yield the titlecompound as a pale yellow foam (4.58 g).

LCMS (System B): t_(RET)=1.08-1.09 min; MH⁺468

¹H NMR (400 MHz, DMSO-d₆) includes δ=7.90-7.65 (m, 1 H), 7.05 (s, 1 H),6.93 (s, 2H), 4.70-4.61 (m, 2 H), 3.73 (s, 6 H), 2.75-2.67 (m, 2 H),1.78-1.67 (m, 2 H), 1.40-1.28 (m, 2 H), 0.90 (t, 3 H)

Intermediate 95-Butyl-3-(6-chlorohex-1-yn-1-yl)-N-(3,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

A nitrogen degassed stirred mixture of5-butyl-N-(3,4-dimethoxybenzyl)-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-amine(4.58 g, 9.7 mmol), copper(I) iodide (278 mg, 1.46 mmol) andbis(triphenylphosphine)palladium(II)dichloride (515 mg, 0.734 mmol) inanhydrous N,N-dimethylformamide (105 mL) was warmed to 60° C. under anitrogen atmosphere before addition of a solution of 6-chloro-1-hexyne(1.712 g, 14.69 mmol) and triethylamine (2.047 mL, 14.69 mmol) inanhydrous nitrogen degassed N,N-dimethylformamide (15 mL), dropwise over5 minutes. The reaction was stirred at 60° C. for 6 hours. The cooledreaction mixture was evaporated in-vacuo and the resultant oilpartitioned between 1:1 water/brine and ethyl acetate. The organic layerwas separated, passed through a hydrophobic frit and evaporated in-vacuoto yield an orange oil. The oil was dissolved in the minimum volume ofdichloromethane, loaded onto a 330 g silica pre-conditioned cartridgeand purified by chromatography using cyclohexane over 1 column volumefollowed by 0-100% ethyl acetate in cyclohexane gradient over 14 columnvolumes followed by ethyl acetate over 2 column volumes. Appropriatefractions were combined and evaporated in-vacuo to yield the titlecompound as a yellow foam (3.334 g)

LCMS (System B): t_(RET)=1.26, 1.28 min; MH⁺456

¹H NMR (400 MHz, CHLOROFORM-d) δ=6.90 (br. s., 2 H), 6.81-6.70 (m, 1 H),4.77 (br. s., 2 H), 3.94-3.72 (m, 6 H), 3.49 (t, J=6.5 Hz, 2 H), 2.88(t, J=7.8 Hz, 2 H), 2.25 (br. s., 2 H), 1.90-1.71 (m, 4 H), 1.62-1.52(m, 2 H), 1.48-1.31 (m, 2 H), 0.93 (t, J=7.3 Hz, 3 H)

Intermediate 105-Butyl-N-(3,4-dimethoxybenzyl)-3-(6-(pyrrolidin-1-yl)hex-1-yn-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

To a solution of5-butyl-3-(6-chlorohex-1-yn-1-yl)-N-(3,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(3.334 g, 7.31 mmol) in anhydrous acetonitrile (4 mL) was addedtriethylamine (3.06 mL, 21.94 mmol) and pyrrolidine (1.831 mL, 21.94mmol). The solution was stirred at 70° C. for 18 hours. A furtherequivalent of pyrrolidine (0.61 mL, 7.31 mmol) and triethylamine (1.019mL, 7.31 mmol) were added to the reaction mixture. The solution wasstirred at 70° C. for a further 3 hours. The cooled reaction mixture wasevaporated in-vacuo and the residue partitioned between ethyl acetateand water/brine (1:1). The organic phase was separated, passed through ahydrophobic frit and evaporated in-vacuo to yield a sticky brown gum(4.226 g). The gum was dissolved in the minimum volume ofdichloromethane, loaded onto a 340 g silica pre-conditioned cartridgeand purified by chromatography using dichloromethane over 1 columnvolume followed by 0-30% methanol (+1% triethylamine) in dichloromethanegradient over 14 column volumes followed by 30% methanol (+1%triethylamine) in dichloromethane over 3 column volumes. Appropriatefractions were combined and evaporated in-vacuo to yield the titlecompound as a sticky brown gum (1.838 g).

LCMS (System B): t_(RET)=1.11-1.17 min; MH⁺491

¹H NMR (400 MHz, METHANOL-d₄) includes δ=7.07-7.04 (m, 1 H), 7.00-6.95(m, 1 H), 6.92 (s, 1 H), 4.76 (s, 2 H), 3.83-3.78 (m, 6 H), 2.82-2.56(m, 10 H), 1.90-1.67 (m, 10 H), 1.44-1.32 (m, 2 H), 0.94 (t, J=7.3 Hz, 3H)

Intermediate 113-(6-Chlorohex-1-yn-1-yl)-5-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

To a nitrogen degassed solution of3-iodo-5-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (307 mg,0.959 mmol) in anhydrous N,N-dimethylformamide (6 mL) under a nitrogenatmosphere at room temperature was added copper(I) iodide (36.5 mg,0.192 mmol), bis(triphenylphosphine)palladium(II)dichloride (75 mg,0.107 mmol) and finally triethylamine (0.267 mL, 1.918 mmol). Themixture was stirred at room temperature under nitrogen atmosphere for 10minutes and then a solution of 6-chlorohex-1-yne (224 mg, 1.918 mmol) inanhydrous N,N-dimethylformamide (1.5 mL) was added. The reaction mixturewas stirred at 60° C. for 2 hours. Another 2 equivalents of6-chlorohex-1-yne (224 mg, 1.918 mmol) and the reaction was left to stirat 60° C. for 1 hour. The reaction was evaporated in-vacuo to yield adark red oil. The oil was partitioned between water/brine (1:1) andethyl acetate. The organic layer was separated, passed through ahydrophobic frit and evaporated in-vacuo to yield an orange oil (621mg). The crude material was dissolved in a minimum amount of DCM, loadedonto a 50 g silica cartridge and purified by chromatography using a0-100% ethyl acetate in cyclohexane gradient over 60 minutes.Appropriate fractions containing desired product were combined andevaporated in-vacuo to yield the title compound as a pale yellow solid(163.5 mg).

LCMS (System A): t_(RET)=0.80 min; MH⁺309, 311

Intermediate 125-(2-Methoxyethyl)-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

To a solution of3-(6-chlorohex-1-yn-1-yl)-5-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one(84 mg, 0.230 mmol) in anhydrous N,N-dimethylformamide (4 mL) was addedpyrrolidine (0.132 mL, 1.579 mmol) and triethylamine (0.293 mL, 2.105mmol). The reaction was stirred at 80° C. for 2 hours. A further 66 μL(1.5 eq) of pyrrolidine and 147 μL (2 eq) of triethylamine were added tothe reaction and the reaction was stirred at 80° C. for 2 hours. Afurther 66 μL (1.5 eq) of pyrrolidine and 147 μL (2 eq) of triethylaminewere added to the reaction. The reaction was stirred at 80° C. for 1hours. The reaction was evaporated in-vacuo to yield a dark yellow oil.A solution of the oil in ethanol (35 mL) was passed through the H-cube(settings: 55° C., full hydrogen, 1 mL/min flow rate and 10% palladiumon carbon CatCart30 as the catalyst). A new 10% palladium on carbonCatCart30 cartridge was inserted into the H-cube and the solution waspassed through the H-cube again (settings: 55° C., full hydrogen, 1mL/min flow rate). The solution was evaporated in-vacuo to yield a paleyellow oil and the crude product was purified by MDAP (Method B).Appropriate fractions were combined and evaporated in-vacuo to give thetitle compound as a clear oil (84 mg).

LCMS (System A): t_(RET)=0.46 min; MH⁺348

Intermediate 135-Butyl-3-(5-chloropent-1-yn-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

To a nitrogen degassed solution of5-butyl-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (250 mg, 0.786mmol) in anhydrous N,N-dimethylformamide (6 mL) under nitrogenatmosphere at room temperature was added copper(I) iodide (30 mg, 0.158mmol), bis(triphenylphosphine)palladium(II)dichloride (62 mg, 0.088mmol) and finally triethylamine (0.219 mL, 1.572 mmol). The mixture wasstirred at room temperature under nitrogen atmosphere for 10 minutes andthen a solution of 5-chloro-1-pentyne (161 mg, 1.572 mmol) in anhydrousN,N-dimethylformamide (1.5 mL) was added. The reaction mixture wasstirred at 60° C. for 80 minutes. The reaction was evaporated in-vacuoto yield a brown oil. The oil was partitioned between water/brine (1:1)and ethyl acetate. The organic layer was separated, passed through ahydrophobic frit and evaporated in-vacuo to yield a brown solid. Thesolid was absorbed onto Florisil, loaded onto a 50 g silica cartridgeand purified by chromatography using a 0-100% ethyl acetate incyclohexane gradient over 60 minutes. Appropriate fractions werecombined and evaporated in-vacuo to yield the title compound as a paleyellow solid (172 mg).

LCMS (System A): t_(RET)=0.91 min; MH⁺293, 295

Intermediate 145-Butyl-3-(5-(piperidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

To a solution of5-butyl-3-(5-chloropent-1-yn-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one(168 mg, 0.574 mmol) in anhydrous N,N-dimethylformamide (3.5 mL) wasadded a solution of piperidine (147 mg, 1.722 mmol) in anhydrousN,N-dimethylformamide (0.5 mL) and triethylamine (0.32 mL, 2.295 mmol).The reaction was stirred at 80° C. for 190 minutes. A further 73 mg(0.857 mmol) of piperidine and 160 μL (1.148 mmol) of triethylamine wasadded to the reaction. The reaction was stirred at 80° C. for a further2.5 hours and then at room temperature for 15.5 hours. A further 73 mg(0.857 mmol) of piperidine and 160 μL (1.148 mmol) of triethylamine wasadded to the reaction. The reaction was stirred at 80° C. for 2.5 hours.The reaction was evaporated in-vacuo to yield a dark yellow oil. Asolution of the oil in ethanol (50 mL) was passed through the H-cube(settings: 30° C., full hydrogen, 1 mL/min flow rate and 10% palladiumon carbon CatCart30 as the catalyst). A new 10% palladium on carbonCatCart30 cartridge was inserted into the H-cube and the solution waspassed through the H-cube again (settings: 30° C., full hydrogen, 1mL/min flow rate). The solution was evaporated in-vacuo to yield a paleyellow oil. The oil was dissolved in MeOH:DMSO (1:1) (3×1 mL) andpurified by MDAP (Method B). Appropriate fractions were combined andevaporated in-vacuo to yield the title compound as a pale yellow solid(91 mg).

LCMS (System B): t_(RET)=0.83 min; MH⁺346

Intermediate 155-Butyl-3-(5-(pyrrolidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6h)-one

To a degassed solution of5-butyl-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7(6M-one (79 mg, 0.248 mmol)in anhydrous N,N-dimethylformamide (2.0 mL) under nitrogen atmosphere atroom temperature was added copper(I) iodide (10 mg, 0.053 mmol),tetrakis(triphenylphosphine)-palladium(0) (32 mg, 0.028 mmol) andfinally triethylamine (0.076 mL, 0.546 mmol). The mixture was stirred atroom temperature under nitrogen atmosphere for 10 minutes and then asolution of 1-(4-pentyn-1-yl)pyrrolidine (75 mg, 0.546 mmol) (ChemicalCommunications 46(19), 3351-3353; 2010) in anhydrousN,N-dimethylformamide (0.5 mL) was added. The reaction mixture wasstirred at 55° C. for 1 hour. A solution of 1-(4-pentyn-1-yl)pyrrolidine(75 mg, 0.546 mmol) in anhydrous N,N-dimethylformamide (0.5 mL) wasadded to the reaction. The reaction mixture was stirred at 55° C. for 40minutes. The reaction was evaporated in-vacuo to yield a dark yellowoil. A filtered solution of the oil in ethanol (15 mL) was passedthrough the H-cube (settings: 45° C., full hydrogen, 1 mL/min flow rateand 10% palladium on carbon CatCart30 as the catalyst). The solution waspassed through the H-cube a further two times using a new CatCart 30cartridge on each occasion. The solution was evaporated in-vacuo toyield a colourless oil. The oil was dissolved in MeOH:DMSO (1:1) (1 mL)and purified by MDAP (Method B). Appropriate fractions were combined andevaporated in-vacuo to yield the title compound as a white solid (20mg).

LCMS (System B): t_(RET)=0.79 min; MH⁺332

Intermediate 165-Butyl-N-(2,4-dimethoxybenzyl)-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-amine

A solution of 5-butyl-3-iodo-1/1-pyrazolo[4,3-d]pyrimidin-7(6/0-one (50g, 157 mmol), (2,4-dimethoxyphenyl)methanamine (60 g, 359 mmol) and2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (47.9 g, 314 mmol) inacetonitrile (500 mL) was treated with((1H-benzo[d][1,2,3]triazol-1-yl)oxy)tri(pyrrolidin-1-yl)phosphoniumhexafluorophosphate(V) (100 g, 192 mmol) and stirred for 6 hours at roomtemperature under nitrogen. The resulting suspension was filtered toremove precipitate and the filtrate was evaporated. The residue waspurified on a silica cartridge (1500 g) (applied in minimum DCM) elutedwith 0-80% cyclohexane-EtOAc (12 column volumes). Product-containingfractions were part evaporated to give a heavy suspension that wasfiltered and the solid air-dried to give the title compound (37.5 g, 80mmol) as a white powder.

LCMS (System A): t_(RET)=0.91 min; MH⁺468

¹H NMR (400 MHz, CHLOROFORM-d) includes δ=6.43-6.33 (m, 2H), 4.76 (s,2H), 3.79 (s, 3H), 3.67 (s, 3H), 2.90-2.80 (m, 2H), 1.84 (s, 2H),1.48-1.35 (m, 2H), 0.94 (t, J=7.3 Hz, 3H)

The mother liquors and impure fractions were evaporated to give yellowsolids. The combined impure materials were triturated with ethyl acetateto give a cream solid (30 g). Further trituration with diethyl ether (50mL) followed by DCM/Et₂O (1;1, 30 mL) gave an additional portion of thetitle compound as a white solid (25.7 g).

Intermediate 175-Butyl-3-(6-chlorohex-1-yn-1-yl)-N-(2,4-dimethoxybenzyl)-1H-Pyrazolo[4,3-d]pyrimidin-7-amine

A solution of5-butyl-N-(2,4-dimethoxybenzyl)-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-amine(50 g, 107 mmol), 6-chloro-1-hexyne (18.71 g, 160 mmol), triethylamine(22.37 mL, 160 mmol), bis(triphenylphosphine)palladium(II)dichloride(5.63 g, 8.02 mmol) and copper(I) iodide (3.04 g, 15.94 mmol) inN,N-Dimethylformamide (1000 mL) under nitrogen was heated to 70° C. for4 hours. The cooled solution was evaporated and the residue was purifiedby chromatography on a 1.5 kg silica cartridge eluted with 20-80%cyclohexane-EtOAc during 12 column volumes. Appropriate fractions wereevaporated to give the title compound as a yellow foam (25.5 g).

LCMS (System A): t_(RET)=1.05 min; MH⁺456, 458

¹H NMR (400 MHz, CHLOROFORM-d) includes δ=6.71-6.54 (m, 1H), 6.41-6.31(m, 2H), 4.75 (br. s., 2H), 3.77 (s, 4H), 3.67 (br. s., 3H), 3.50-3.43(m, 2H), 2.89-2.76 (m, 3H), 2.26-2.15 (m, 2H), 1.30-1.20 (m, 1H), 0.92(t, J=7.3 Hz, 3H)

Intermediate 185-Butyl-N-(2,4-dimethoxybenzyl)-3-(6-(pyrrolidin-1-yl)hex-1-yn-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

A solution of5-butyl-3-(6-chlorohex-1-yn-1-yl)-N-(2,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(30 g, 65.8 mmol), pyrrolidine (16.32 mL, 197 mmol) and Et₃N (27.5 mL,197 mmol) in Acetonitrile (400 mL) was heated at 70° C. for 16 hours.Further pyrrolidine (7 g) was added and the solution was heated at 70°C. for 8 hours. The cooled solution was evaporated and the residue waspurified on a silica cartridge (750 g) eluted with EtOAc (2 columnvolumes) followed by 20-30% MeOH (+1% Et₃N)/EtOAc (16 column volumes) togive the title compound as a yellow gum (23.3 g).

LCMS (System B): t_(RET)=1.15 min; MH⁺491

¹H NMR (400 MHz, CHLOROFORM-d) includes δ=7.31-7.25 (m, 1H), 6.42-6.23(m, 2H), 4.76 (br. S., 2H), 3.76 (s, 3H), 3.65 (s, 3H), 2.88-2.78 (m,6H), 2.67-2.60 (m, 2H), 2.18-2.11 (m, 2H_, 1.93-1.75 (m, 6H), 1.63 (br.S., 2H), 1.43-1.28 (m, 4H), 0.91 (t, J=7.3 Hz, 3H).

Intermediate 195-Butyl-N-(2,4-dimethoxybenzyl)-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

A solution of5-butyl-N-(2,4-dimethoxybenzyl)-3-(6-(pyrrolidin-1-yl)hex-1-yn-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(20 g, 40.8 mmol) in ethanol (500 mL) was hydrogenated over Pd—C (5 g,4.70 mmol) for 4 hour (H₂ uptake complete). The mixture was filteredthrough Hyflo and evaporated. The residue in DCM (30 mL) was purified ona 120 g silica cartridge (Redisep Gold) eluted with toluene-ethanol-NH₃(85/15/1.5) in 7 batches—taking off product and lower running impurityand recycling the cartridge each time (total volume ˜3 liters).Evaporation of appropriate fractions gave the title compound as a yellowgum (16.3 g).

LCMS (System B): t_(RET)=1.15 min; MH⁺495

¹H NMR (400 MHz, CHLOROFORM-d) includes δ•=6.49-6.41 (m, 2H), 6.12-6.02(m, 1H), 4.82 (br. s., 2H), 3.85-3.78 (m, 6H), 3.04-2.96 (m, 2H),2.88-2.80 (m, 2H), 2.57 (br. s., 4H), 2.50-2.43 (m, 2H), 0.97 (t, ••=7.3Hz, 3H)

Intermediate 207-(5-Butyl-7((2,4-dimethoxybenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)heptan-1-ol

Bis(triphenylphosphine)palladium(II)dichloride (68 mg, 0.097 mmol),triethylamine (0.537 mL, 3.85 mmol) and copper iodide (37 mg, 0.194mmol) were added to a nitrogen degassed solution of5-butyl-N-(2,4-dimethoxybenzyl)-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-amine(600 mg, 1.284 mmol) in anhydrous N,N-dimethylformamide (15 mL). Themixture was stirred under a nitrogen atmosphere and heated to 60° C.before the addition of hept-6-yn-1-ol (432 mg, 3.85 mmol) in anhydrousN,N-dimethylformamide (2 mL). The reaction was stirred at 60° C. for 2.5hours. An additional 0.5 equivalents of hept-6-yn-1-ol (72 mg, 0.642mmol) in anhydrous N,N-dimethylformamide (0.5 mL) was added to thereaction. The mixture was stirred for a further 2.5 hours under anitrogen atmosphere at 60° C. A further 0.5 equivalents ofhept-6-yn-1-ol (72 mg, 0.642 mmol) in anhydrous N,N-dimethylformamide(0.5 mL) was added to the reaction. The mixture was stirred for afurther 4 hours under a nitrogen atmosphere at 60° C. and then allowedto cool to ambient temperature overnight. The reaction mixture wasevaporated in-vacuo to yield a brown oil. The resulting oil waspartitioned between ethyl acetate (25 mL) and water/brine (1:1, 15 mL).The organic layer was separated and the aqueous phase back-extractedwith ethyl acetate (25 mL). The combined organic extracts were passedthrough a hydrophobic frit and evaporated in-vacuo to yield theintermediate alkyne as an orange oil (1.21 g).

A mixture of the oil and 10% palladium on carbon (200 mg) in ethanol (20mL) was stirred under a hydrogen atmosphere for 22 hours. The reactionwas filtered through a celite cartridge (10 g) and evaporated in-vacuoto yield an orange oil. The oil was dissolved in the minimum volume ofdichloromethane, loaded onto a 50 g silica cartridge and purified usinga 0-100% ethyl acetate in cyclohexane gradient over 60 minutes followedby elution with ethyl acetate (300 mL) then 10% methanol indichloromethane (70 mL). Appropriate fractions were combined andevaporated in vacuo to yield an orange oil. The oil was dissolved in theminimum volume of dichloromethane, loaded onto a 50 g silica cartridgeand purified using a 0-10% methanol in dichloromethane gradient over 40mins. (detection wavelength=230 nm). Appropriate fractions were combinedand evaporated in vacuo to yield a yellow oil (380 mg). A mixture of theoil and 10% palladium on carbon (200 mg) in ethanol (20 mL) was stirredunder a hydrogen atmosphere for 20 hours. The reaction was filteredthrough a celite cartridge (10 g) and evaporated in-vacuo to yield thetitle compound as a pale yellow oil (264 mg).

LCMS (System B): t_(RET)=1.17 min; MH⁺456

Intermediate 213-(7-Bromoheptyl)-5-butyl-N-(2,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

A solution of triphenylphosphine (182 mg, 0.695 mmol) in dichloromethane(2 mL) was added dropwise to a stirred solution of7-(5-butyl-7-((2,4-dimethoxybenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)heptan-1-ol(264 mg, 0.579 mmol) and carbon tetrabromide (231 mg, 0.695 mmol) indichloromethane (5 mL). The reaction was stirred at room temperature for18 hours. An additional portion of carbon tetrabromide (231 mg, 0.695mmol) and triphenylphosphine (182 mg, 0.695 mmol) were added to thereaction mixture. Stirring at ambient temperature was continued for 2hours. The solvent was evaporated in-vacuo to yield a yellow oil. Theoil was dissolved in anhydrous dichloromethane (DCM) (7 mL) and stirredat room temperature for 1.5 hours. Carbon tetrabromide (231 mg, 0.695mmol) and triphenylphosphine (182 mg, 0.695 mmol) were added to thereaction mixture. Stirring at ambient temperature was continued for 17.5hours. Additional carbon tetrabromide (115 mg, 0.348 mmol) andtriphenylphosphine (91 mg, 0.348 mmol) were added to the reactionmixture. Stirring at ambient temperature was continued for 2 hours. Thereaction was evaporated in-vacuo to yield a clear oil.

The resulting oil was partitioned between ethyl acetate (10 mL) andwater/brine (1:1, 15 mL). The organic layer was separated, passedthrough a hydrophobic frit and evaporated in-vacuo to yield a paleyellow solid (1.40 g). The solid was dissolved in the minimum volume ofdichloromethane, loaded onto a 50 g silica cartridge and purified usinga 0-100% ethyl acetate in cyclohexane gradient over 60 minutes, followedby 0-20% methanol in DCM over 30 minutes. Fractions containing only onecomponent were combined and evaporated in-vacuo to yield the titlecompound as a pale yellow oil (55 mg).

LCMS (System B): t_(RET)=1.47 min; MH⁺518, 520

Fractions containing two components were combined and evaporatedin-vacuo to yield an additional portion of the title compound as a 1:1mixture with triphenyphosphine oxide as a pale yellow oil (270 mg)

LCMS (System B): t_(RET)=1.47 min; MH⁺518, 520: 0.98 min; MH⁺279

Intermediate 223-(6-(Azepan-1-yl)hexyl)-5-butyl-N-(2,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

To a suspension of5-butyl-3-(6-chlorohex-1-yn-1-yl)-N-(2,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(400 mg, 0.877 mmol) and hexamethyleneimine (0.297 mL, 2.63 mmol) inacetonitrile (7 mL) was added triethylamine (0.367 mL, 2.63 mmol) atambient temperature. The reaction mixture was stirred at 60° C. undernitrogen for 16 hours. The temperature was increased to 80° C. and thereaction mixture was left to stir under nitrogen for a further 7 hours.To the reaction mixture was added a solution of hexamethyleneimine(0.149 mL, 1.32 mmol) and triethylamine (0.184 mL, 1.32 mmol) and wasleft to stir under nitrogen 60° C. for a further 72 hours. The reactionmixture was evaporated in vacuo to give an brown oil which waspartitioned between ethyl acetate (50 mL) and water (50 mL). The aqueouslayer was extracted and the organic layer was washed with brine (50 mL),dried using a hydrophobic frit and concentrated in vacuo to give a brownoil. The oil was dissolved in minimal amount of dichloromethane, whichwas then loaded and purified on aminopropyl silica cartridge (50 g)using a 0-100% ethyl acetate-cyclohexane gradient followed by a 0-25%methanol-dichloromethane gradient for 60 minutes. The appropriatefractions were combined and evaporated in vacuo to give a brown oil,which was then azeotroped with dichloromethane and petroleum ether(40-60) to give a brown solid (0.185 g). The solid was dissolved inethanol (45 mL) and was hydrogenated using the H-cube (settings: 40° C.,Full H₂, 1 mL/min flow rate) and 5% Pd/C CatCart 30 as the catalyst. Thesolvent obtained was concentrated in vacuo to give a brown oil which wasdissolved in ethanol (20 mL) and was hydrogenated using the H-cube(settings: 40° C., Full H₂, 1 mL/min flow rate) and 5% Pd/C CatCart 30as the catalyst. The solvent was further hydrogenated using the H-cube(settings: 40° C., Full H₂, 1 mL/min flow rate) and the same 5% Pd/CCatCart 30 as the catalyst. The solution obtained was evaporated invacuo to give a brown oil. The brown oil was then dissolved in minimalamount of dichloromethane, which was then loaded and purified bychromatography on aminopropyl silica (20 g) using a 0-10%methanol-dichloromethane gradient for 40 minutes. The appropriatefractions were combined and evaporated in vacuo to give a brown oil,which was then azeotroped with dichloromethane and petroleum ether(40-60) to give the title compound as a brown oil (0.100 g).

LCMS (System B): t_(RET)=1.19 min; MH⁺523

Intermediate 235-Butyl-3-(5-chloropent-1-yn-1-yl)-N-(2,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

Bis(triphenylphosphine)palladium(II)dichloride (101 mg, 0.144 mmol),triethylamine (0.403 mL, 2.89 mmol) and copper iodide (55.0 mg, 0.289mmol) were added to a nitrogen degassed solution of5-butyl-N-(2,4-dimethoxybenzyl)-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-amine(900 mg, 1.926 mmol) in anhydrous N,N-dimethylformamide (23 mL). Themixture was stirred under a nitrogen atmosphere and heated to 60° C.before the addition of 5-chloropent-1-yne (296 mg, 2.89 mmol) inanhydrous N,N-dimethylformamide (2 mL). The reaction was stirred at 60°C. for 3 hours. A further 0.30 equivalents of 5-chloropent-1-yne (59 mg,0.575 mmol) in anhydrous N,N-dimethylformamide (1 mL) was added to thereaction. The mixture was stirred at 60° C. for a further 2.5 hours andthen allowed to cool to room temperature overnight. The reaction mixturewas evaporated in-vacuo to yield a brown oil. The resulting oil waspartitioned between ethyl acetate (25 mL) and water/brine (1:1, 10 mL).The organic layer was separated, passed through a hydrophobic frit andevaporated in-vacuo to yield a brown oil (1.32 g). The oil was dissolvedin the minimum volume of dichloromethane, loaded onto a 100 g silicacartridge and purified by chromatography using a 0-100% ethyl acetate incyclohexane gradient over 60 minutes. Appropriate fractions werecombined and evaporated in-vacuo to yield an orange oil. The oil wasdissolved in the minimum volume of dichloromethane, loaded onto a 100 gsilica cartridge and re-purified by chromatography using a 0-100% ethylacetate in cyclohexane gradient over 60 minutes. Appropriate fractionswere combined and evaporated in-vacuo to yield the title compound as anorange oil (720 mg).

LCMS (System B): t_(RET)=1.32 min; MH⁺442, 444

Intermediate 24(S)-5-Butyl-N-(3,4-dimethoxybenzyl)-3-(6-(3-fluoropyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

To a stirred solution of5-butyl-3-(6-chlorohex-1-yn-1-yl)-N-(3,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(225 mg, 0.493 mmol) in acetonitrile (4 mL) was added triethylamine(0.413 mL, 2.96 mmol) and (S)-3-fluoropyrrolidine hydrochloride (186 mg,1.480 mmol). The resultant mixture was heated at 60° C. for 22 hours. Tothe reaction mixture was added further (S)-3-fluoropyrrolidinehydrochloride (186 mg, 1.480 mmol) and further triethylamine (0.413 mL,2.96 mmol) and heating at 75° C. continued for 20 hours. The reactionmixture was concentrated in vacuo and the residue partitioned betweenDCM (20 mL) and water (20 mL). The organic was separated and dried usinga hydrophobic frit before concentration in vacuo to give a dark red oil.The crude material was dissolved in MeOH (40 mL) and hydrogenated usingthe H-Cube (setting: 40° C., full H₂). The crude mixture wasconcentrated in vacuo, re-dissolved in a minimum amount of DCM andloaded on the top of an aminopropyl silica cartridge (70 g). The columnwas eluted using a gradient 0-100% EtOAc/cyclohexane over 60 minutesfollowed by flushing with 0-25% MeOH/DCM. The appropriate fractions werecombined and evaporated in vacuo to give an orange oil. The oil wasdissolved in methanol (40 mL) and run through the H-cube (setting: fullH₂, 40° C., 10% palladium on carbon CatCart30 as the catalyst). Thesolution was concentrated in vacuo to yield the title compound as paleyellow oil (119 mg).

LCMS (System B): t_(RET)=1.19 min; MH⁺513

Intermediate 25(S)-5-Butyl-N-(2,4-dimethoxybenzyl)-3-(5-(3-fluoropyrrolidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

Triethylamine (0.860 mL, 6.20 mmol) was added to a mixture of(S)-3-fluoropyrrolidine hydrochloride (390 mg, 3.10 mmol) and5-butyl-3-(5-chloropent-1-yn-1-yl)-N-(2,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(457 mg, 1.034 mmol) in N,N-dimethylformamide (7 mL). The mixture washeated to 80° C. and stirred for 18 hours. An additional portion of(S)-3-fluoropyrrolidine hydrochloride (195 mg, 1.55 mmol) andtriethylamine (0.430 mL, 3.10 mol) were added to the reaction mixture.The reaction was stirred at 80° C. for a further 6 hours.N,N-Dimethylformamide (3 mL) was added and the reaction mixture wasstirred at 80° C. for a further 18 hours and then allowed to cool toroom temperature. The mixture was evaporated in-vacuo to yield a brownoil. The resulting oil was partitioned between dichloromethane (50 mL)and water/brine (20 mL). The organic layer was separated, passed througha hydrophobic frit and evaporated in-vacuo to yield a brown oil. The oilwas dissolved in the minimum volume of dichloromethane, loaded onto a 50g aminopropyl functionalised silica cartridge and purified using a 0-10%methanol in dichloromethane gradient over 40 minutes. (detectionwavelength=230 nm). Appropriate fractions were combined and evaporatedin-vacuo to yield a brown oil (261 mg). A mixture of the oil and 10%palladium on carbon (50 mg) in ethanol (20 mL) was stirred under ahydrogen atmosphere for 3 hours. Additional 10% palladium on carbon (50mg) was added to the reaction and the mixture was stirred under ahydrogen atmosphere for 18 hours. A further portion of 10% palladium oncarbon (50 mg) was added to the reaction and the mixture was stirredunder a hydrogen atmosphere for 5 hours. The reaction was filteredthrough a celite cartridge (10 g) and evaporated in-vacuo to yield abrown oil. The oil was dissolved in the minimum volume ofdichloromethane, loaded onto a 50 g aminopropyl functionalised silicacartridge and purified using a 0-10% methanol in dichloromethanegradient over 40 mins. (detection wavelength=230 nm). Appropriatefractions were combined and evaporated in-vacuo to yield the titlecompound as a brown oil (175 mg).

LCMS (System B): t_(RET)=1.23 min; MH⁺499

Intermediate 26(R)-5-Butyl-N-(3,4-dimethoxybenzyl)-3-(6-(3-fluoropyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

Prepared similarly to Intermediate 24 from5-butyl-3-(6-chlorohex-1-yn-1-yl)-N-(3,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amineand (R)-3-fluoropyrrolidine hydrochloride

LCMS (System B): t_(RET)=1.17 min; MH⁺513

Intermediate 27(R)-5-butyl-N-(2,4-dimethoxybenzyl)-3-(5-(3-fluoropyrrolidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

Prepared similarly to Intermediate 25 from and5-butyl-3-(5-chloropent-1-yn-1-yl)-AA(2,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine and(R)-3-fluoropyrrolidine hydrochloride

LCMS (System B): t_(RET)=1.23 min; MH⁺499

Intermediate 281-(6-(5-Butyl-74(2,4-dimethoxybenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)hexyl)piperidin-4-ol

Prepared similarly to Intermediate 22 from5-butyl-3-(6-chlorohex-1-yn-1-yl)-N-(2,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amineand 4-hydroxypiperidine

LCMS (System B): t_(RET)=1.09 min; MH⁺525

Intermediate 291-(5-(5-Butyl-7-((2,4-dimethoxybenzyl)amino)-1H-Pyrazolo[4,3-d]pyrimidin-3-yl)pentyl)piperidin-4-ol

Triethylamine (0.327 mL, 2.355 mmol) was added to a solution of4-hydroxypiperidine (238 mg, 2.355 mmol) and5-butyl-3-(5-chloropent-1-yn-1-yl)-N-(2,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(347 mg, 0.785 mmol) in anhydrous N,N-Dimethylformamide (5 mL). Thesolution was heated to 80° C. and stirred for 17 hours. An additionalportion of 4-hydroxypiperidine (79 mg, 0.785 mmol) was added to thereaction mixture and stirring was continued at 80° C. for 1 hour. Thereaction mixture was evaporated in-vacuo to yield a brown oil. Theresulting oil was partitioned between dichloromethane (2×25 mL) andwater/brine (1:1, 20 mL). The organic layer was separated, passedthrough a hydrophobic frit and evaporated in-vacuo to yield a brown oil(643 mg). The oil was dissolved in the minimum volume ofdichloromethane, loaded onto a 50 g aminopropyl silica cartridge andpurified using a 0-10% methanol in dichloromethane gradient over 40minutes (detection wavelength=230 nm). Appropriate fractions werecombined and evaporated in-vacuo to yield a brown solid, (253 mg). Amixture of the solid and 10% palladium on carbon (100 mg) in ethanol (20mL) was stirred under a hydrogen atmosphere for 6 hours. An additionalportion of 10% palladium on carbon (100 mg) was added to the reactionand the mixture was stirred under hydrogen for a further 16 hours. Afurther portion of 10% palladium on carbon (50 mg) was added to thereaction and the mixture was stirred under hydrogen for a further 3hours. The reaction was filtered through a celite cartridge (10 g) andevaporated in-vacuo to yield the title compound as a brown oil (224 mg).

LCMS (System B): t_(RET)=1.07 min; MH⁺511

Intermediate 305-Butyl-N-(3,4-dimethoxybenzyl)-3-(6-(4-fluoropiperidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

Prepared similarly to Intermediate 24 from5-butyl-3-(6-chlorohex-1-yn-1-yl)-N-(3,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amineand 4-fluoropiperidine hydrochloride

LCMS (System B): t_(RET)=1.23 min; MH⁺527

Intermediate 315-Butyl-N-(2,4-dimethoxybenzyl)-3-(5-(4-fluoropiperidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

Prepared similarly to Intermediate 25 from and5-butyl-3-(5-chloropent-1-yn-1-yl)-N-(2,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amineand 4-fluoropiperidine hydrochloride LCMS (System B): t_(RET)=1.26 min;MH⁺513

EXAMPLE PREPARATION Example 15-Butyl-3-(6-(Piperidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amineformate

A mixture of5-butyl-3-(6-(piperidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one(87 mg, 0.242 mmol) and phosphorus oxychloride (1.5 mL, 16.09 mmol) washeated at 120° C. for 45 minutes. The reaction was cooled to roomtemperature and added dropwise with vigorous stirring to 20% aqueoussodium hydroxide solution (24 mL) (pH of mixture after addition was 14).The mixture was adjusted to pH12 using 2M aqueous citric acid solutionand extracted into ethyl acetate. The organic layer was separated,passed through a hydrophobic frit and evaporated in-vacuo to yield ared/brown oil (95 mg). The material (95 mg) was suspended in 2-propanol(2 mL) and 35% (0.88) ammonia solution (2 mL). The reaction was stirredat 140° C. for 90 minutes in a Biotage Initiator microwave. Reactionevaporated in-vacuo to yield a red oil. The oil was dissolved inMeOH:DMSO (1:1) (2×1 mL) and purified by MDAP (Method A). Theappropriate fractions were combined and evaporated in-vacuo to yield thetitle compound as a colourless oil (13.2 mg).

LCMS (System A): t_(RET)=0.44 min; MH⁺359

Example 25-(2-Methoxyethyl)-3-(6-(piperidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

A mixture of5-(2-methoxyethyl)-3-(6-(piperidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one(116.5 mg, 0.322 mmol) and phosphorus oxychloride (1.9 mL, 20.38 mmol)was heated at 120° C. for 45 minutes. The reaction was cooled to roomtemperature and evaporated in-vacuo to yield a red/brown oil. Toluene (5mL) was added to the oil and the resulting suspension was evaporatedin-vacuo to yield a red/brown oil. The oil was dissolved in iso-propanol(2 mL) and 0.88 ammonia (2 mL, 36.2 mmol) was added. The reaction wasstirred at 100° C. for 60 minutes in a Biotage Initiator microwave thethe reaction was evaporated in-vacuo to yield a red solid. The oil wasdissolved in MeOH:DMSO (1:1) (2×1 mL) and purified by MDAP (Method B).Appropriate fractions were combined and evaporated in-vacuo to yield thetitle compound as a clear oil (15.6 mg).

LCMS (System B): t_(RET)=0.76 min; MH⁺361

Example 35-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

Method A

A mixture of5-butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one(245 mg, 0.709 mmol) and phosphorus oxychloride (12.89 mL, 138 mmol) wasstirred at 120° C. for 45 mins. The reaction was cooled to roomtemperature and evaporated in-vacuo to yield a red/brown oil. Toluene(10 mL) was added to the oil and the resulting suspension was evaporatedin-vacuo to yield a red/brown oil. The oil was dissolved in iso-propanol(7 mL) and 0.88 ammonia (5.88 mL, 106 mmol) was added. The reaction wasstirred at 120° C. for 60 minutes in a Biotage Initiator microwave thenthe reaction evaporated in-vacuo to yield an orange solid. The crudematerial was purified by reverse phase chromatography using MDAP (MethodA). Fractions which contained product were evaporated in-vacuo to yieldthe title compound as a colourless oil (72.1 mg).

LCMS (System B): t_(RET)=0.84 min; MH⁺345

Method B

A mixture of5-butyl-N-(3,4-dimethoxybenzyl)-3-(6-(pyrrolidin-1-yl)hex-1-yn-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(1.838 g, 3.75 mmol) and 10 wt % palladium on carbon (200 mg) in ethanol(53 mL) was stirred under a hydrogen atmosphere at room temperature for2 hours. A further 300 mg of 10 wt % palladium on carbon was added tothe reaction under a nitrogen atmosphere and the reaction was stirredunder a hydrogen atmosphere at room temperature for 2 hours. A further250 mg of 10 wt % palladium on carbon was added to the reaction under anitrogen atmosphere and the reaction was stirred under a hydrogenatmosphere at room temperature for 16 hours. The reaction mixture wasfiltered through a 10 g celite cartridge and the filtrate evaporatedin-vacuo to yield a sticky brown oil (2.2 g) The oil was dissolved intrifluoroacetic acid (10 mL) and heated in a Biotage Initiator microwave(using initial absorption setting high) to 120° C. for 4 hours then wasleft at room temperature for 17 hours. The black/very dark greenreaction mixture was evaporated in-vacuo and the remaining residuepartitioned between 25% 2-propanol in chloroform (500 mL) and 0.1Maqueous sodium hydroxide solution (500 mL). The organic phase was passedthrough a hydrophobic frit and evaporated in-vacuo to yield a brown gum(1.74 g). The gum was dissolved in the minimum volume ofdichloromethane, loaded onto a 100 g dichloromethane pre-conditionedsilica cartridge and purified by chromatography using dichloromethaneover 1 column volume followed by 0-30% methanol (+1% triethylamine) indichloromethane gradient over 18 column volumes followed by 30% methanol(+1% triethylamine) in dichloromethane over 6 column volumes (detectionwavelength=230 nm). Appropriate fractions were combined and evaporatedin-vacuo to yield the title compound as a pale brown solid (596 mg).

LCMS (System A): t_(RET)=0.80 min; MH⁺345

Method C

5-butyl-N-(2,4-dimethoxybenzyl)-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(5.33 g, 10.77 mmol) was dissolved in trifluoroacetic acid (45 mL, 584mmol) at ambient temperature. The reaction mixture was stirred at 60° C.for 3 h. The reaction mixture was evaporated in vacuo and the residuewas dissolved in 3:1 chloroform:IPA (500 mL) and washed with dilute (0.2M) aqueous sodium hydroxide (400 mL). The aqueous mixture was extractedwith further portions of 3:1 chloroform:IPA (2×400 mL). The combinedorganic phase was filtered through a hydrophobic frit and the filtratewas evaporated in vacuo to give a yellow solid (4.7 g). A portion of thecrude material (1.26 g) was dissolved in dichloromethane/methane(1519:1) and the cloudy solution filtered through a pad of glassfibrefilter papers under vacuum. The filtrate was evaporated in vacuo and theresidue triturated with ether. The suspension was filtered and the solidmaterial washed with ether to give the title compound as an off-whitesolid (0.695 g).

LCMS (System B): t_(RET)=0.83 min; MH⁺345

¹H NMR (400 MHz, METHANOL-d₄) δ=3.02-2.92 (m, 6H), 2.87-2.80 (m, 2H),2.78-2.71 (m, 2H), 1.95 (s, 4H), 1.85-1.71 (m, 4H), 1.68-1.58 (m, 2H),1.49-1.35 (m, 6H), 0.96 (t, J=7.3 Hz, 3H)

The remainder of the material was dissolved in dichloromethane/methane(50 mL, 19:1) and the cloudy solution filtered through a pad ofglassfibre filter papers under vacuum. The filtrate was evaporated invacuo and the residue triturated with ether. The suspension was filteredand the solid material washed with ether to give a further potion of thetitle compound as an off-white solid (2.174 g).

LCMS (System B): t_(RET)=0.85 min; MH⁺345

Example 45-(2-Methoxyethyl)-3-(6-(Pyrrolidin-1-yl)hexyl)-1H-Pyrazolo[4,3-d]pyrimidin-7-amine

Prepared similarly to Example 2 from5-(2-methoxyethyl)-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

LCMS (System B): t_(RET)=0.65 min; MH⁺347

Example 55-Butyl-3-(5-(Piperidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

Prepared similarly to Example 2 from5-butyl-3-(5-(piperidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one.

LCMS (System B): t_(RET)=0.84 min; MH⁺345

Example 65-Butyl-3-(5-(pyrrolidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

Prepared similarly to Example 2 from5-butyl-3-(5-(pyrrolidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

LCMS (System B): t_(RET)=0.84 min; MH⁺345

Example 75-Butyl-3-(7-(Piperidin-1-yl)heptyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

Triethylamine (0.085 mL, 0.608 mmol) was added to a solution of3-(7-bromoheptyl)-5-butyl-N-(2,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(150 mg, 0.203 mmol) and piperidine (0.060 mL, 0.608 mmol) in anhydrousacetonitrile (7 mL). The solution was stirred at ambient temperature for1.5 hours. Additional piperidine (0.060 mL, 0.608 mmol) andtriethylamine (0.085 mL, 0.608 mmol) were added to the reaction mixtureand stirring at ambient temperature was continued for 17 hours. Thereaction mixture was evaporated in vacuo to yield a clear oil. The oilwas partitioned between ethyl acetate (20 mL) and water/brine (1:1, 5mL). The organic layer was separated, passed through a hydrophobic fritand evaporated in vacuo to yield a clear oil. The oil was dissolved intrifluoroacetic acid (3 mL, 38.9 mmol) and heated at 60° C. for 3 hours.The reaction mixture was evaporated in-vacuo and the residue wasdissolved in 3:1 chloroform:IPA (20 mL) and washed with aqueous sodiumhydroxide (0.1M, 5 mL). The organic phase was separated using ahydrophobic frit and the aqueous back extracted with 3:1 chloroform:IPA(20 mL). The combined organic extracts were evaporated in-vacuo to yielda yellow oil (215 mg). The oil was dissolved in MeOH:DMSO (1:1) (3×1 mL)and purified by MDAP (Method B). Appropriate fractions were combined andevaporated in-vacuo to yield a brown oil (88.2 mg). The oil wasdissolved in MeOH:DMSO (1:1) (1 mL) and purified by MDAP (Method A).Appropriate fractions were combined and evaporated in-vacuo to yield thetitle compound as a white solid (40.5 mg).

LCMS (System B): t_(RET)=0.94 min; MH⁺373

Example 85-Butyl-3-(7-(pyrrolidin-1-yl)heptyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

Prepared similarly to Example 7 from3-(7-bromoheptyl)-5-butyl-N-(2,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amineand pyrrolidine

LCMS (System B): t_(RET)=0.82 min; MH⁺359

Example 93-(6-(Azepan-1-yl)hexyl)-5-butyl-1H-Pyrazolo[4,3-d]pyrimidin-7-amine

3-(6-(Azepan-1-yl)hexyl)-5-butyl-N-(2,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(0.100 g, 0.191 mmol) was dissolved in trifluoroacetic acid (2 mL, 26.0mmol) at ambient temperature. The reaction mixture was stirred at 60° C.for 4 hours. The reaction mixture was evaporated in vacuo and theresidue was dissolved in 3:1 chloroform:IPA (15 mL) and washed withaqueous sodium hydroxide (0.1 M, 5 mL). The organic phase was separatedusing a hydrophobic frit and evaporated in vacuo to give a brown oil(0.157 g). The oil was dissolved in MeOH:DMSO (1:1) (2 mL) and purifiedby MDAP (Method B). Appropriate fractions were combined and evaporatedin-vacuo to yield the title compound as an off-white solid (38 mg).

LCMS (System B): t_(RET)=0.88 min; MH⁺373

Example 103-(5-(AzePan-1-yl)pentyl)-5-butyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine

Triethylamine (0.282 mL, 2.036 mmol) was added to a solution ofhexamethyleneimine (0.229 mL, 2.036 mmol) and5-butyl-3-(5-chloropent-1-yn-1-yl)-N-(2,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(300 mg, 0.679 mmol) in anhydrous N,N-Dimethylformamide (5 mL). Thesolution was heated to 80° C. and stirred for 3.5 hours. A further 1.5equivalents of hexamethyleneimine (101 mg, 1.018 mmol) and triethylamine(0.141 mL, 1.018 mmol) in anhydrous N,N-dimethylformamide (2 mL) wereadded to the reaction mixture and stirring continued at 80° C. for 18hours. An additional portion of hexamethyleneimine (101 mg, 1.018 mmol)and triethylamine (0.141 mL, 1.018 mmol) in anhydrousN,N-dimethylformamide (3 mL) was added to the reaction mixture andstirring continued at 80° C. for 5.5 hours. The reaction mixture wasevaporated in-vacuo to yield a brown oil. The resulting oil waspartitioned between dichloromethane (25 mL) and water/brine (1:1, 20mL). The organic layer was separated, passed through a hydrophobic fritand evaporated in-vacuo to yield a brown oil. The oil was dissolved inthe minimum volume of dichloromethane, loaded onto a 50 g aminopropylsilica cartridge and purified using a 0-10% methanol in dichloromethanegradient over 40 minutes (detection wavelength=230 nm). Appropriatefractions were combined and evaporated in-vacuo to yield a brown oil,(235 mg). The oil was dissolved in the minimum volume ofdichloromethane, loaded onto a 50 g aminopropyl silica cartridge andre-purified using a 0-10% methanol in dichloromethane gradient over 40mins. (detection wavelength=230 nm). Appropriate fractions were combinedand evaporated in-vacuo to yield the intermediate alkyne as a brown oil,(195 mg). A mixture of the oil and 10% palladium on carbon (150 mg) inethanol (20 mL) was stirred under a hydrogen atmosphere for 19 hours.Additional 10% palladium on carbon (100 mg) was added to the reactionand the mixture was stirred under a hydrogen atmosphere for a further 23hours. Additional 10% palladium on carbon (100 mg) was added to thereaction and the mixture was stirred under a hydrogen atmosphere for afurther 5 hours. The reaction was filtered through a celite cartridge(10 g) and evaporated in-vacuo to yield as a brown oil (180 mg). The oilwas dissolved in MeOH:DMSO (1:1) (3 mL) and purified by MDAP (Method B).Appropriate fractions were combined and evaporated in-vacuo to yield ayellow oil.

The oil was dissolved in trifluoroacetic acid (3 mL, 38.9 mmol) andheated at 60° C. for 3 hours. The reaction was stirred at 60° C. for afurther 2 hours and then allowed to cool to room temperature over night.The reaction mixture was evaporated in-vacuo and the residue wasdissolved in 3:1 chloroform:IPA (15 mL) and washed with aqueous sodiumhydroxide (0.1M, 6 mL). The organic phase was separated using ahydrophobic frit and the aqueous back extracted with 3:1 chloroform:IPA(15 mL). The combined organic extracts were evaporated in-vacuo to yielda brown oil. The oil was dissolved in MeOH:DMSO (1:1) (3 mL) andpurified by MDAP (Method B). Appropriate fractions were combined andevaporated in-vacuo to yield the title compound as an pale beige solid(32 mg).

LCMS (System B): t_(RET)=0.79 min; MH⁺359

Example 11(S)-5-Butyl-3-(6-(3-fluoropyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

A solution of(S)-5-butyl-N-(3,4-dimethoxybenzyl)-3-(6-(3-fluoropyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(119 mg, 0.232 mmol) in trifluoroacetic acid (1 mL, 12.98 mmol) washeated in a Biotage Initiator microwave (using initial absortion settinghigh) at 120° C. for 2.5 h. The reaction mixture was concentrated invacuo and the residue was dissolved in 3:1 chloroform:IPA (50 mL) andwashed with aqueous sodium hydroxide (0.1 M, 50 mL). The organic phasewas separated and the aqueous back extracted with 3:1 chloroform:IPA (30mL). The combined organic extracts were dried (hydrophobic frit) andconcentrated in vacuo. The material (223 mg) was dissolved in MeOH:DMSO(1:1) (2 mL) and purified by MDAP (Method B). Appropriate fractions werecombined and evaporated in-vacuo to yield the title compound as anorange solid (33 mg).

LCMS (System B): t_(RET)=0.91 min; MH⁺363

Example 12(S)-5-Butyl-3-(5-(3-fluoropyrrolidin-1-yl)pentyl)-1H-Pyrazolo[4,3-d]pyrimidin-7-amine

Prepared similarly to Example 9 from(S)-5-butyl-N-(2,4-dimethoxybenzyl)-3-(5-(3-fluoropyrrolidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

LCMS (System B): t_(RET)=0.85 min; MH⁺349

Example 13(R)-5-Butyl-3-(6-(3-fluoropyrrolidin-1-yl)hexyl)-1H-Pyrazolo[4,3-d]pyrimidin-7-amineformate

Prepared similarly to Example 11 from(R)-5-butyl-N-(3,4-dimethoxybenzyl)-3-(6-(3-fluoropyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

LCMS (System B): t_(RET)=0.88 min; MH⁺363

Example 14(R)-5-Butyl-3-(5-(3-fluoropyrrolidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

Prepared similarly to Example 9 from(R)-5-butyl-N-(2,4-dimethoxybenzyl)-3-(5-(3-fluoropyrrolidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

LCMS (System B): t_(RET)=0.85 min; MH⁺349

Example 151-(6-(7-Amino-5-butyl-1H-pyrazolo[4,3-d]pyrimidin-3-yl)hexyl)piperidin-4-ol

Prepared similarly to Example 9 from1-(6-(5-butyl-7-((2,4-dimethoxybenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)hexyl)piperidin-4-ol

LCMS (System B): t_(RET)=0.76 min; MH⁺375

Example 161-(5-(7-Amino-5-butyl-1H-pyrazolo[4,3-d]pyrimidin-3-yl)pentyl)piperidin-4-ol

Prepared similarly to Example 9 from1-(5-(5-butyl-7-((2,4-dimethoxybenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)pentyl)piperidin-4-ol

LCMS (System B): t_(RET)=0.71 min; MH⁺361

Example 175-Butyl-3-(6-(4-fluoropiperidin-1-yl)hexyl)-1H-Pyrazolo[4,3-d]pyrimidin-7-amineformate

Prepared similarly to Example 11 from5-butyl-N-(3,4-dimethoxybenzyl)-3-(6-(4-fluoropiperidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

LCMS (System B): t_(RET)=0.93 min; MH⁺377

Example 185-Butyl-3-(5-(4-fluoropiperidin-1-yl)pentyl)-1H-Pyrazolo[4,3-d]pyrimidin-7-amine

Prepared similarly to Example 9 from5-butyl-N-(2,4-dimethoxybenzyl)-3-(5-(4-fluoropiperidin-1-yl)pentyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine

LCMS (System B): t_(RET)=0.90 min; MH⁺363

Example 195-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-Pyrazolo[4,3-d]pyrimidin-7-aminemaleate

A round bottomed flask charged with5-butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(140 mg, 0.406 mmol) was treated with IPA (1.4 mL, 10 vol) and stirredunder a nitrogen atmosphere at 50° C. until a homogeneous solution wasgenerated. Maleic acid (47.2 mg, 0.406 mmol) was added directly as asolid and the resulting reaction mixture stirred till completedissolution of maleic acid had occurred. The resulting solution was thentreated with a sonicated slurry of5-butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-aminemaleate (0.5 mg) in IPA (0.1 mL) and the resulting slurry was aged at50° C. for 1 hour before being allowed to cool to 20° C. over 4 hours.The resulting slurry was then stirred overnight at 20° C. The slurry wascooled to 3° C. in an ice bath and aged for 30 minutes prior to thesolid being collected via vacuum filtration. The resulting solid waswashed with chilled IPA (2×1 mL) before being oven dried at 40° C. invacuo overnight to yield an off white solid (122 mg). The resultingsolid was further dried in vacuo at 40° C. to afford the title compoundas an off-white solid (95 mg).

LCMS (System B): t_(RET)=0.79 min; MH⁺345

¹H NMR (400 MHz, DMSO-d₆) includes δ=7.05 (br. s, 2H), 6.02 (s, 2H),3.13-3.00 (m, 3H), 2.83 (br. s., 2H), 2.64 (br. s., 2H), 2.03-1.83 (m,4H), 1.80-1.53 (m, 6H), 1.43-1.25 (m, 6H), 0.90 (t, J=7.5 Hz, 3H)

Example 205-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-Pyrazolo[4,3-d]pyrimidin-7-aminehemimaleate

5-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(205 mg,) was treated with maleic acid (34.5 mg, 0.5 eq) before beingslurried in IPA (2.0 mL). The resulting reaction mixture was heated to70° C. resulting in a homogeneous solution which on aging at 70° C.started to precipitate solid. The resulting slurry was found to setsolid after 30 mins at 70° C. therefore additional IPA (0.5 mL) wasadded to mobilize the slurry. The resulting reaction mixture was allowedto cool to 20° C. and was aged overnight at 20° C. before being filteredand washed with fresh IPA (2×4 mL) to yield a white solid which was ovendried at 40° C. in vacuo (90 mbar) overnight. The resulting solid (199mg) was further dried in vacuo at 50° C. to afford the title compound.

¹H NMR (400 MHz, DMSO-d₆) includes δ=7.05 (br. s, 2H), 6.01 (s, 1H),1.89-1.61 (m, 8H), 1.56-1.43 (m, 2H), 1.41-1.24 (m, 6H), 0.89 (t, J=7.4Hz, 3H)

Example 215-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-Pyrazolo[4,3-d]pyrimidin-7-aminedimaleate

5-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(197.3 mg,) was treated with maleic acid (133 mg, 2.0 eq) before beingslurried in IPA (2.0 mL). The resulting reaction mixture was heated to70° C. resulting in a homogeneous solution which on aging at 70° C.started to precipitate solid. The resulting slurry was found to setsolid at 70° C. therefore an additional IPA (2.0 mL) was added tomobilise the slurry. The resulting reaction mixture was allowed to coolslowly to 20° C. and was aged overnight at 20° C. before being filteredand washed with fresh IPA (2×4 mL) to yield a white solid which was ovendried at 40° C. in vacuo (90 mbar) overnight to afford the titlecompound (294 mg).

¹H NMR (400 MHz, DMSO-d₆) includes δ=6.07 (s, 4H), 3.15-3.03 (m, 2H),2.80-2.64 (m, 2H), 2.09-1.51 (m, J=7.9 Hz, 10H), 1.42-1.25 (m, 6H), 0.91(t, J=7.4 Hz, 3H)

Example 225-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-Pyrazolo[4,3-d]pyrimidin-7-aminehemi-succinate

5-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(196.7 mg,) was treated with succinic acid (33.7 mg, 0.5 eq) beforebeing slurried in IPA (2.0 mL). The resulting reaction mixture washeated to 70° C. resulting in a homogeneous solution which on aging at70° C. started to precipitate solid. The resulting slurry was found toset solid at 70° C. therefore additional IPA (1.0 mL) was added tomobilize the slurry.

The resulting reaction mixture was allowed to cool slowly to 20° C. andwas aged overnight at 20° C. before being filtered and washed with freshIPA (2×4 ml) to yield a white solid which was oven dried at 40° C. invacuo (90 mbar) overnight. The resulting solid (193 mg) was furtherdried in vacuo at 50° C. to afford the title compound.

¹H NMR (400 MHz, DMSO-d₆) includes δ=7.08 (br. s., 2H), 2.88-2.75 (m,2H), 2.32 (s, 2H), 1.80-1.59 (m, 8H), 1.53-1.22 (m, 8H), 0.89 (t, J=7.4Hz, 3H)

Example 235-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-aminemethanesulfonate

5-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(200.3 mg,) slurried in acetonitrile (2 mL) at 70° C. was treated withanhydrous methane sulfonic acid (38 ul, 1.0 eq). The resulting reactionmixture turned over to a solution on addition of methane sulfonic acidand on aging at 70° C. started to precipitate solid. The resultingslurry was found to set solid and therefore additional acetonitrile (2mL) was added to mobilize the slurry. The resulting reaction mixture wasallowed to cool to 20° C. and was aged overnight at 20° C. before beingfiltered and washed with fresh acetonitrile (2×4 mL) to yield a whitesolid which was oven dried at 40° C. in vacuo (90 mbar) overnight toafford the title compound (234 mg).

¹H NMR (400 MHz, DMSO-d₆) includes δ=7.17-6.94 (m, 2H), 2.89-2.76 (m,2H), 2.64 (br. s., 2H), 2.32 (s, 3H), 1.68 (s, 6H), 1.43-1.23 (m, 6H),0.90 (t, J=7.4 Hz, 3H)

Example 245-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-aminehydrochloride

5-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(197.2 mg) was slurried in acetonitrile (2 mL) at 70° C. before treatedwith 1.25M HCl in IPA (0.458 mL) which generated a solution. On aging at70° C. the reaction started to precipitate a solid and this stayedmobile throughout age at 70° C. and therefore the reaction was cooled to20° C. The resulting slurry was aged overnight at 20° C. before beingfiltered and washed with fresh acetonitrile (2×4 mL) to yield a whitesolid which was oven dried at 40° C. in vacuo (90 mbar) overnight. Theresulting solid (144 mg) was further dried in vacuo at 50° C. to affordthe title compound.

¹H NMR (400 MHz, DMSO-d₆) includes δ=7.18 (br. s, 2H), 2.72-2.57 (m,2H), 2.04-1.54 (m, 10H), 1.42-1.23 (m, 6H), 0.90 (t, J=7.4 Hz, 3H)

Polymorphism

X-Ray Powder Diffraction (XRPD) was performed on5-butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amineaccording to the following methods.

XRPD

The data were acquired on a PANalytical X'Pert Pro powderdiffractometer, model PW3040/60 using an X'Celerator detector. Theacquisition conditions were: radiation: Cu Kα, generator tension: 40 kV,generator current: 45 mA, start angle: 2.0° 2θ, end angle: 40.0° 2θ,step size: 0.0167° 2θ, time per step: 31.75 seconds. The sample wasprepared by mounting a few milligrams of sample on a silicon wafer (zerobackground) plate, resulting in a thin layer of powder.

Characteristic XRPD angles and d-spacings for batches are recorded inTable 1 for Examples 21 and 22. The margin of error is approximately±0.1° 2θ for each of the peak assignments. Peak intensities may varyfrom sample to sample due to preferred orientation.

Peak positions were measured using Highscore software.

TABLE 1 Characteristic XRPD Peak Positions for Examples 21 and 22Example 21 Example 22 2θ/° d-spacings/Å 2θ/° d-spacings/Å 5.3 16.8 8.110.9 5.8 15.2 9.8 9.1 6.4 13.9 11.6 7.6 9.0 9.8 16.0 5.5 10.1 8.8 17.55.1 10.9 8.1 19.5 4.5 11.6 7.7 20.2 4.4 12.7 7.0 23.0 3.9 16.0 5.5 23.73.7 19.1 4.7

Representative XRPD diffractograms are shown in FIG. 1 and FIG. 2.

BIOLOGICAL EVALUATION

Compounds of the invention were tested for in vitro biological activityin accordance with the following assay.

Assay for the Induction of Interferon-α• and TNF-α Using Fresh HumanWhole Blood (WB)

Compound Preparation

Compounds were prepared at 100× required concentration in DMSO inflat-bottom microtitre plates at a volume of 1.5 μL. Columns 1-10contained a 1 in 4 serial dilution of the test compound. Included oneach plate was a serial dilution of the TLR7/8 agonist resiquimod as astandard and Column 11 contained 1.5 μl of 200 μM resiquimod (giving a 2μM final concentration, used to define the approximate maximal responseto resiquimod). Each compound was assayed in duplicate for each donor.

Incubation and Assays for Interferon-α• and TNF-α

Blood samples from three human donors were collected into sodium heparin(10 U/ml). 150 μl of whole Blood was dispensed into Col 1 to 11 of assayplates containing 1.5 μl of test compound or standard in DMSO. Plateswere placed in an incubator overnight (37° C., 95% air, 5% CO₂).Following the overnight incubation, plates were removed from theincubator & mixed on an orbital shaker for approximately 1 minute. 100μl of 0.9% saline was added to each well and the plates mixed again onan orbital shaker. Plates were then centrifuged (2500 rpm, 10 mins),after which a sample of plasma was removed using a Biomek FX and assayedfor both IFN-α and TNF-α using the MSD (Mesoscale Discovery)electrochemiluminescence assay platform. The IFN-α assay was carried outsimilarly to that described above. The TNF-α assay was carried out asper kit instructions (Cat No K111BHB).

Cytokine released was expressed as a percentage of the 2 μM resiquimodcontrol (column 11). This percentage was plotted against compoundconcentration and the pEC₅₀ for the response determined by non-linearleast squares curve fitting. For the IFN-α responses, generally a 4parameter logistic model was selected. For the TNF responses where aclear maximum response was obtained (i.e. a well defined plateau in theresponse was observed) then a 4 parameter model was generally used. Ifthe upper asymptote of the curve wasn't well defined then the curvefitting was generally constrained to a maximal response of 100% (i.e. tothe response to 2 μM resiquimod) or to the response of the highestconcentration tested if this was greater than the resiquimod response.Some curves were bell shaped for one or both cytokines and the cytokinedata on the down slope of the bell shaped response (i.e. concentrationsabove those giving the maximal response) were generally excluded fromthe fit, usually with the exception of the concentration immediatelyabove the peak response. Curve fitting thus concentrated on the up slopeof the dose response curve.

Results

Examples 1 to 24 had a mean pEC₅₀ for IFNα• of ≧5.9. Examples 3, 21 and22 had mean pEC₅₀ for IFNα of 6.9, 7.2 and 7.6 respectively.

Examples 1 to 24 had a mean pEC₅₀ for TNF-α of ≧5.4. Examples 3, 21 and22 had mean pEC₅₀ for THF-α of 4.7, <4.3 and 4.7 respectively.

The invention claimed is:
 1. A method of treatment of a disease orcondition selected from: asthma and allergic rhinitis, which methodcomprises administering to a human subject in need thereof, atherapeutically effective amount of the compound5-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine, of formula:

or a salt thereof.
 2. A method of treatment according to claim 1,wherein the salt is a pharmaceutically acceptable salt.
 3. A method oftreatment according to claim 2, wherein said compound or salt is5-Butyl-3-(6-(pyrrolidin-1-yl)hexyl)-1 H-pyrazolo[4,3-d]pyrimidin-7-amine maleate.
 4. A method of treatment according toclaim 1, wherein said compound or salt is in the form of a free base. 5.A method of treatment according to claim 1 wherein said disease orcondition is allergic rhinitis.
 6. A method of treatment according toclaim 2 wherein said disease or condition is allergic rhinitis.
 7. Amethod of treatment according to claim 3 wherein said disease orcondition is allergic rhinitis.
 8. A method of treatment according toclaim 4 wherein said disease or condition is allergic rhinitis.
 9. Amethod of treatment according to claim 1 wherein said disease orcondition is asthma.
 10. A method of treatment according to claim 2wherein said disease or condition is asthma.
 11. A method of treatmentaccording to claim 3 wherein said disease or condition is asthma.
 12. Amethod of treatment according to claim 4 wherein said disease orcondition is asthma.